Haptens, hapten conjugates, compositions thereof preparation and method for their preparation and use

ABSTRACT

A method for performing a multiplexed diagnostic assay, such as for two or more different targets in a sample, is described. One embodiment comprised contacting the sample with two or more specific binding moieties that bind specifically to two or more different targets. The two or more specific binding moieties are conjugated to different haptens, and at least one of the haptens is an oxazole, a pyrazole, a thiazole, a nitroaryl compound other than dinitrophenyl, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azo aryl, or a benzodiazepine. The sample is contacted with two or more different anti-hapten antibodies that can be detected separately. The two or more different anti-hapten antibodies may be conjugated to different detectable labels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/660,017, filed Feb. 17, 2010, which is a divisional of U.S. patentapplication Ser. No. 11/982,627, filed Nov. 1, 2007, which claims thebenefit of U.S. Provisional Application No. 60/856,133, filed Nov. 1,2006. The entire disclosures of these prior applications areincorporated herein by reference.

FIELD

This disclosure concerns haptens, hapten conjugates, and diagnostic andtherapeutic compositions thereof. More particularly, this disclosureconcerns haptens, hapten conjugates and anti-hapten antibody conjugatesthat can be utilized in various combinations for the simultaneousidentification, visualization and/or quantitation of a plurality oftargets in a sample, such as multiple protein and nucleic acid targetsin a tissue sample.

BACKGROUND

Generally, only large molecules, infectious agents, and insolubleforeign matter can elicit an immune response in an animal. However,haptens, which are small molecules, can in certain instances be inducedto elicit an immune response if they are first coupled to a largecarrier (such as a protein) to form an immunogen. Haptens in combinationwith anti-hapten antibodies that are raised against the immunogens andisolated are useful for detecting particular molecular targets. Forexample, specific binding moieties such as primary antibodies andnucleic acid probes can be labeled with one or more hapten molecules,and once these specific binding moieties are bound to their moleculartargets they can be detected using an anti-hapten antibody conjugatethat includes a detectable label such as an enzyme or a fluorescentlabel. Binding of the detectable anti-hapten antibody conjugate to asample indicates the presence of the target in a sample.

Digoxigenin, present exclusively in Digitalis plants as a secondarymetabolite, is an example of a hapten that has been utilized in avariety of molecular assays. U.S. Pat. No. 4,469,797, entitled“Digoxigenin Immunogens, Antibodies, Label Conjugates and RelatedDerivatives,” discloses using immunoassays to determine digoxinconcentrations in blood samples based upon the specific binding ofantidigoxin antibodies to the drug in the test sample. U.S. Pat. No.5,198,537, entitled “Digoxigenin Derivatives and Use Thereof,” describesa number of additional digoxigenin derivatives that have been used inimmunological tests, such as immunoassays.

Other haptens have been developed for analytical procedures includingbiotin and fluorescein. However, each of these haptens has specificdrawbacks that have made dioxigenin the hapten of choice for sensitiveimmunoassays. In the case of biotin, certain biological samples includeendogenous biotin that can lead to background interference. Similarly,fluorescein, a fluorescent molecule, can lead to background fluorescencein a fluorescent immunoassay. For in situ assays such asimmunohistochemical (IHC) assays and in situ hydridization (ISH) assaysof tissue and cytological samples, especially multiplexed assays of suchsamples, it is highly desirable to identify and develop new haptens andanti-hapten antibodies (and conjugates thereof) to provide additionalassay flexibility, especially since it is becoming clear that samplescan best be characterized through simultaneous detection of multipletargets.

A primary goal of cancer therapy is to selectively kill, or inhibituncontrolled growth of, malignant cells while not adversely affectingnormal cells. Traditional chemotherapeutic drugs are highly cytotoxic,and while preferably having greater affinity for malignant cells thanfor normal cells, nevertheless typically adversely affect normal cells.New therapeutics are now being developed that target the growth factorand nutrient pathways that regulate cell growth and metabolism inresponse to intracellular and environmental cues. These signalingpathways often are altered or dysregulated in cancer. For example,certain growth factors (such as EGF, a growth factor that activatesprotein-receptor tyrosine kinase (“RTK”) activity to initiate a signaltransduction cascade resulting in changes in cell growth, proliferationand differentiation) are involved in the pathogenesis and progression ofdifferent cancers. Such pathways and associated signaling moleculesprovide attractive targets for therapeutic intervention, but it isbecoming increasingly evident that different populations of patientshave tumors that appear to be dysregulated in different manners. Forexample, a particular therapeutic target (or combination of therapeutictargets) may only be present in tumors from certain populations ofpatients, and thus identifying such certain populations having thetarget (or combination of targets) can be used to stratify patients intopotential non-responders and potential non-responders to a therapeutic(or combination of therapeutics) directed toward the target (ortargets). The use of companion diagnostics to stratify patients in thismanner is a first step toward personalizing the treatment of cancer inindividual patients. Increased individualization of treatments willcertainly involve multiplexed assays for multiple therapeutic targets.

Unfortunately, in recent years there has been little research directedto developing additional classes of haptens against which sensitive andspecific antibodies can be raised in order to enable highly multiplexedassays. Such highly multiplexed assays would be useful for monitoringthe response of individuals to a given therapeutic regimen and forcompanion diagnostic applications. Identifying additional classes ofhaptens and methods for their use in analytical and therapeuticapplications would substantially advance the state of the art in thisfield.

SUMMARY

Thus, based on the above, a need exists in the art for additionalhaptens, and hapten conjugates, that are useful for diagnostic and/ortherapeutic applications. Accordingly, certain disclosed embodiments ofthe present invention concern new classes of haptens, hapten conjugatesand compositions thereof.

Embodiments of a method for performing a multiplexed diagnostic assay,such as for two or more different targets in a sample, are described.One embodiment comprised contacting the sample with two or more specificbinding moieties that bind specifically to two or more differenttargets. The two or more specific binding moieties are conjugated todifferent haptens, and at least one of the haptens is an oxazole, apyrazole, a thiazole, a nitroaryl compound, a benzofurazan, atriterpene, a urea, a thiourea, a rotenoid, a coumarin or a cyclolignan.The sample is contacted with two or more different anti-haptenantibodies that can be detected separately. The two or more differentanti-hapten antibodies may be conjugated to different detectable labels.In some embodiments, the two or more different anti-hapten antibodiesare from different mammalian species. The method may further comprisecontacting the two or more different anti-hapten antibodies with two ormore anti-antibodies that specifically bind the two or more differentanti-hapten antibodies. For such embodiments, the two or moreanti-antibodies may be conjugated to different detectable labels.

Certain embodiments of the hapten are azoles having the followinggeneral chemical formula

where R₁-R₄ independently are selected from hydrogen, acyl, aldehydes,alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oximeether, alcohols, amido, amino, amino acid, aryl, alkyl aryl,carbohydrate, monosaccharides, disaccharides, oligosaccharides,polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester,ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl,hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl,sulfoxide, and combinations thereof, at least one of the R₁-R₄substituents being bonded to a linker or is a reactive group suitablefor coupling to a linker or a carrier molecule, X independently isnitrogen or carbon, Y is oxygen, sulfur or nitrogen, and if Y is oxygenor sulfur, then there is no R₁ group, and if Y is nitrogen, then thereis at least one R₁ group. One specific example of such a hapten has thefollowing structure.

Another class of haptens is the nitroaryl compounds having the followinggeneral chemical formula

where at least one of R₁-R₆ is nitro, and the remaining R₁-R₆ ringsubstituents independently are selected from hydrogen, acyl, aldehydes,alkoxy, aliphatic heteroaliphatic, oxime, oxime ether, alcohols, amido,amino, amino acid, aryl, alkyl aryl, carbohydrate, monosaccharides,disaccharides, oligosaccharides, polysaccharides, carbonyl, carboxyl,carboxylate, cyclic, heterocyclic, cyano, ester, ether, halogen,heteroaryl, hydroxyl, hydroxlyamine, keto, sulfhydryl, sulfonyl,sulfoxide, exomethylene, or two or more of the R₁-R₆ substituents areatoms in a ring system, and at least one of the R₁-R₆ substituents isbonded to a linker or is a reactive group suitable for coupling to thelinker.

Another class of haptens is the benzofurazans or derivatives thereof,such as compounds having a formula

where the R₁-R₄ substituents independently are selected from hydrogen,acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic,heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid,aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides,oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate,cyclic, cyano, ester, ether, exomethylene, halogen, heteroaryl,heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro,sulfhydryl, sulfonyl, sulfoxide, and combinations thereof, or two ormore of the R₁-R₄ substituents are atoms in a ring system bonded orfused to the compounds having the illustrated general formula, at leastone of the R₁-R₄ substituents being bonded to the linker or is thereactive group, and Y is oxygen, sulfur or a carbon atom having R₅ andR₆ substituents, where R₅ and R₆ are as stated for R₁-R₄. One specificexample of such a hapten has the following structure.

Another class of haptens is cyclic terpenes having a formula

where R₁-R₂₁ independently are selected from hydrogen, acyl, aldehydes,alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oximeether, alcohols, amido, amino, amino acid, aryl, alkyl aryl,carbohydrate, monosaccharides, disaccharides, oligosaccharides,polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester,ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl,hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl,sulfoxide, and combinations thereof, at least one of the R₁-R₂₁substituents being bonded to a linker or is a reactive group suitablefor coupling to the linker or a carrier molecule, and where two or moreof R₁-R₂₁ substituents may be atoms in a ring system bonded or fused tothe compounds having the illustrated general formula, Y is a bond,thereby defining a 5-membered ring, or is a carbon atom bearing R₂₂ andR₂₃ substituents, where R₂₂ and R₂₃ are as stated for R₁-R₂₁. Onespecific example of such a hapten has the following structure.

Another class of haptens is ureas and thioureas having a formula

where R₁-R₃ independently are hydrogen, aliphatic, substitutedaliphatic, cyclic, heterocyclic, aryl and heteroaryl, and Y is oxygen orsulfur. Particular examples of ureas or thioureas are aryl ureas orthioureas having a formula

where R₁-R₇ independently are independently are selected from hydrogen,acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic,heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid,aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides,oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate,cyclic, cyano, ester, ether, exomethylene, halogen, heteroaryl,heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro,sulfhydryl, sulfonyl, sulfoxide, and combinations thereof, at least oneof the R₁-R₇ substituents is bonded to a linker or is a reactive group,and where two or more of R₁-R₇ substituents may be atoms in a ringsystem bonded or fused to the compounds having the illustrated generalformula, and Y is oxygen or sulfur. One specific example of such ahapten has a formula

Another class of haptens is the rotenoids having a formula

where R₁-R₁₄ independently are hydrogen, acyl, aldehydes, alkoxy,aliphatic, substituted aliphatic, heteroaliphatic, oxime, oxime ether,alcohols, amido, amino, amino acid, aryl, alkyl aryl, carbohydrate,monosaccharides, disaccharides, oligosaccharides, polysaccharides,carbonyl, carboxyl, carboxylate, cyclic, cyano, ester, ether,exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl,hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl,sulfoxide, and combinations thereof, at least one of the R₁-R₁₄substituents is coupled to a linker or is a reactive group, and wheretwo or more of R₁-R₁₄ substituents may be atoms in a ring system bondedor fused to the compounds having the illustrated general formula. Onespecific example of such a hapten has a formula

The rotenone haptens also include rotenone isoxazolines, which typicallyhave a formula

With reference to the rotenone isoxazolines, R-R₅ independently arehydrogen, aldehyde, alkoxy, aliphatic, particularly lower aliphatic,including all branched chain isomers, such as isoprene, and allstereoisomers, substituted aliphatic, heteroaliphatic, e.g., organicchains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl,particularly alkyl having 20 or fewer carbon atoms, and even moretypically lower alkyl having 10 or fewer atoms, such as methyl, ethyl,propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide(e.g. —CX₃ where X is a halide, and combinations thereof, either in thechain or bonded thereto) amino, amino acid, amido, cyano (—CN), halogen,hydroxyl, hydroxylamine, oxime (HO—N═), oxime ether (e.g., methoxyimine,CH₃—O—N═) alkyl hydroxyl, particularly lower alkyl hydroxyl, carbonyl,keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide,carboxyl, carboxylate (and salts thereof, such as Group I metal orammonium ion carboxylates) ester, alkyl ester, acyl, exomethylene,ether, cyclic, heterocyclic, aryl, alkyl aryl, such as benzyl,heteroaryl, polysaccharides, carbohydrate, monosaccharides, such asglucose and fructose, disaccharides, such as sucrose and lactose,oligosaccharides and polysaccharides, and combinations thereof. At leastone of the R-R₅ substituents also is bonded to a linker or to a carriermolecule. Y is oxygen, nitrogen, or sulfur.

Another class of haptens is oxazoles or thiazoles having a formula

where R₁-R₃ independently are selected from hydrogen, acyl, aldehydes,alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oximeether, alcohols, amido, amino, amino acid, aryl, alkyl aryl,carbohydrate, monosaccharides, disaccharides, oligosaccharides,polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester,ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl,hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl,sulfoxide, and combinations thereof, at least one of the R₁-R₃substituents is coupled to a linker or is a reactive group, and whereR₂-R₃ substituents may be atoms in a ring system bonded or fused to thecompounds having the illustrated general formula, and Y is oxygen orsulfur. A specific example of such a hapten has the following chemicalstructure.

Another class of haptens is the coumarins or coumarin derivatives havinga formula

where R₁-R₆ independently are selected from hydrogen, acyl, aldehydes,alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oximeether, alcohols, amido, amino, amino acid, aryl, alkyl aryl,carbohydrate, monosaccharides, disaccharides, oligosaccharides,polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester,ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl,hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl,sulfoxide, and combinations thereof, or two or more of the R₁-R₆substituents available for forming such compounds also may be atoms in aring system bonded or fused to the compounds having the illustratedgeneral formula, at least one of the R₁-R₆ substituents is coupled to alinker or is a reactive group, and Y is oxygen, nitrogen or sulfur.

Another class of haptens is the cyclolignans having a formula

where R₁-R₁₂ independently are selected from hydrogen, acyl, aldehydes,alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oximeether, alcohols, amido, amino, amino acid, aryl, alkyl aryl,carbohydrate, monosaccharides, disaccharides, oligosaccharides,polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester,ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl,hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl,sulfoxide, and combinations thereof, or two or more of the R₁-R₁₂substituents available for forming such compounds also may be atoms in aring system bonded or fused to the compounds having the illustratedgeneral formula, at least one of the R₁-R₁₂ substituents is coupled to alinker or is a reactive group. Specific examples of cyclolignan haptensinclude

Another general class of haptens of the present invention isheterobicyclic/biaryl compounds, typically phenyl quinolines andquinoxalines. The heterobicyclic/biaryl compounds typically have a firstgeneral chemical formula as below.

R₁-R₂ substituents independently are selected from: hydrogen, acyl,aldehydes, alkoxy, aliphatic, particularly lower aliphatic, substitutedaliphatic, heteroaliphatic, e.g., organic chains having heteroatoms,such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 orfewer carbon atoms, and even more typically lower alkyl having 10 orfewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl,substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is a halide,and combinations thereof, either in the chain or bonded thereto), oxime,oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic oralkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, aminoacid, aryl, alkyl aryl, such as benzyl, alkoxy aryl, such as methoxy andethoxy, carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. Two or more of the R₁-R₂ substituents, mosttypically plural R₁ substituents, also may be atoms, typically carbonatoms, in a ring system bonded or fused to the compounds having theillustrated general formula. At least one of the R₁-R₂ substituents isbonded to a linker or directly to a carrier. Y is oxygen, nitrogen orsulfur, typically nitrogen. If Y is nitrogen, then the formula also caninclude double bonds to the one or more nitrogen atoms.

Compounds having a single heteroatom are exemplified byphenylquinolines, as follows.

Compounds having two heteroatoms are represented by quinoxalines, asindicated by the general formula below.

Particular examples include2-(3,4-dimethoxyphenyl)quinoline-4-carboxylic acid)

and 3-hydroxy-2-quinoxalinecarbamide.

Another general class of haptens is azoaryl compounds, such asazobenzenes, having a first general chemical formula as below.

R₁-R₂ substituents independently are selected from: hydrogen, acyl,aldehydes, alkoxy, aliphatic, particulary lower aliphatic, substitutedaliphatic, heteroaliphatic, e.g., organic chains having heteroatoms,such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 orfewer carbon atoms, and even more typically lower alkyl having 10 orfewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl,substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is a halide,and combinations thereof, either in the chain or bonded thereto,),oxime, oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e.aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido,amino, amino acid, aryl, alkyl aryl, such as benzyl, alkoxy aryl, suchas methoxy and ethoxy, carbohydrate, monosaccharides, such as glucoseand fructose, disaccharides, such as sucrose and lactose,oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate(including salts thereof, such as Group I metal or ammonium ioncarboxylates), cyclic, heterocyclic, cyano (—CN), ester, alkyl ester,ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═),keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide,sulfonyl, exomethylene, and combinations thereof. Two or more R₂substituents also may be atoms, typically carbon atoms, in a ring systembonded or fused to the compounds having the illustrated general formula.For example, 2 R₂ substituents may form a fused phenyl ring, or a fusedheterocyclic or heteroaryl structure. A particular azoaryl haptens,4-(dimethylamino)azobenzene-4′-sulfonyl chloride, has the formulaprovided below.

Another class of haptens is benzodiazepine having a first generalformula as indicated below.

R₁-R₅ independently are selected from: acyl, aldehydes, alkoxy,aliphatic, particularly lower aliphatic, substituted aliphatic,heteroaliphatic, e.g., organic chains having heteroatoms, such asoxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewercarbon atoms, and even more typically lower alkyl having 10 or feweratoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substitutedalkyl, such as alkyl halide (e.g. —CX₃ where X is a halide, andcombinations thereof, either in the chain or bonded thereto), oxime,oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic oralkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, aminoacid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides,such as glucose and fructose, disaccharides, such as sucrose andlactose, oligosaccharides and polysaccharides, carbonyl, carboxyl,carboxylate (including salts thereof, such as Group I metal or ammoniumion carboxylates), cyclic, cyano (—CN), ester, ether, exomethylene,halogen, heteroaryl, heterocyclic, hydrogen, hydroxyl, hydroxylamine,oxime (HO—N═), keto, such as aliphatic ketones, nitro, sulfhydryl,sulfonyl, sulfoxide, sulfonyl, and combinations thereof. Two or more ofthe R₅ substituents also may be atoms, typically carbon atoms, in a ringsystem bonded or fused to the compounds having the illustrated generalformula. At least one of the R₁-R₅ positions is bonded to a linker or isoccupied by a functional group suitable for coupling to a linker or acarrier molecule. R₁-R₅ most typically are aliphatic, aryl, hydrogen, orhydroxyl, even more typically alkyl, hydrogen or phenyl. Y is oxygen orsulfur, most typically oxygen. A particular example of a benzodiazepinehapten, -(2-hydroxyphenyl)-1H-benzo[b][1,4]diazepine-2(3H)-one, isprovided below.

The present disclosure also provides embodiments of a compound having aformula (hapten)_(m)-(linker)_(n)-(reactive group)_(o) where the haptenis an oxazole, pyrazole, thiazole, nitroaryl, benzofurazan, triterpene,urea, thiourea, rotenoid, coumarin, cyclolignan, or combinationsthereof, m is from 1 to about 200, n is 0 to about 200, and o is from 1to 200. In certain embodiments m is 1 to about 100, n is from o to about5, and o is from about 1 to about 5, and in other embodiments m, n and oare 1.

The present disclosure also describes hapten-carrier conjugatescomprising a hapten coupled to a carrier where the hapten is an oxazole,pyrazole, thiazole, nitroaryl, benzofurazan, triterpene, urea, thiourea,rotenoid, coumarin, cyclolignan, or combinations thereof. Certainembodiments of such conjugates have a formula(hapten)_(m)-(linker)_(n)-(carrier)_(p) where m is from 1 to about 200,n is 0 to about 200 and p is from 1 to about 10. For other embodiments mis from 1 to about 100, n is 1 to 100, and p is from 1 to about 5, andyet other embodiments m, n, o and p are 1. For certain embodiments, thelinker is heteroaliphatic, such as alkyl or alkylene oxides, with oneparticular embodiment comprising an ethylene glycol linker having from 1to about 15 ethylene glycol units. The carrier may be a specific bindingcarrier, such as a protein, a nucleic acid, or an antibody. The carrieralso may be an immunogenic carrier.

The present disclosure also concerns antibodies that specifically bindto a hapten selected from an oxazole, pyrazole, thiazole, nitroaryl,benzofurazan, triterpene, urea, thiourea, rotenoid, coumarin, orcyclolignan.

Pharmaceutical compositions also are described. One embodiment of apharmaceutical composition comprised diagnostically or therapeuticallyeffective amounts of a hapten-carrier conjugate comprising a haptencoupled to a carrier where the hapten is an oxazole, pyrazole, thiazole,nitroaryl, benzofurazan, triterpene, urea, thiourea, rotenoid, coumarin,cyclolignan, or combinations thereof. For such compositions, thehapten-carrier conjugate may have a formula(hapten)_(m)-(linker)_(n)-(reactive group)_(o)-(carrier)_(p) where thehapten is an oxazole, pyrazole, thiazole, nitroaryl, benzofurazan,triterpene, urea, thiourea, rotenoid, coumarin, cyclolignan, orcombinations thereof.

Multiplexed arrays also are described. For example, disclosedembodiments of a multiplexed assay comprised a hapten selected fromoxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes,ureas, thioureas, rotenones, coumarins, cyclolignans, and combinationsthereof.

Kits for use in an enzyme immunoassay are disclosed. Certain kitembodiments comprise a hapten-conjugated antibody or hapten conjugatedto a nucleic acid probe, the hapten being selected from oxazoles,pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas,thioureas, rotenones, coumarins, cyclolignans, and combinations thereof.Such kits also typically include an anti-hapten antibody conjugated to adetectable label.

Embodiments of an immunoassay process are described. For example, theimmunoassay process may comprise providing a reactive hapten conjugatesuitable for performing the immunoassay, the hapten being selected fromoxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes,ureas, thioureas, rotenones, coumarins, cyclolignans, and combinationsthereof. The hapten conjugate is then used in at least one step of theimmunoassay. The hapten conjugate may be a hapten-linker conjugate.Alternatively, the hapten conjugate may be a hapten-carrier conjugate,where the carrier might be an immunogenic carrier or a specific bindingcarrier.

A method for identifying a mammalian tumor is disclosed. One embodimentcomprises assaying a sample obtained from the mammalian tumor to detecta pattern of expression, phosphorylation or both expression andphosphorylation, using a hapten conjugate where the hapten is selectedfrom oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans,triterpenes, ureas, thioureas, rotenones, coumarins, cyclolignans, andcombinations thereof.

A method for assessing a response to drug therapy in an individual alsois disclosed. One embodiment of the method comprised obtaining a firsttissue or cell sample from the individual before exposing the individualto a drug therapy. A second tissue or cell sample is obtained from theindividual after exposing the individual to the drug therapy. Abiochemical product and/or process affected by the therapy is detectedfrom the first sample and the second sample, where detecting comprisesusing a hapten conjugate having a hapten selected from oxazoles,pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas,thioureas, rotenones, coumarins, cyclolignans, and combinations thereof.The results for the first sample are compared to the second sample todetermine whether the drug therapy had a positive, negative or nulleffect.

A method for making a conjugate comprising a hapten also is disclosed.One embodiment of the method comprised providing a hapten selected fromoxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes,ureas, thioureas, rotenones, coumarins, cyclolignans, and combinationsthereof. The hapten is then coupled to a linker or a carrier.

A method for detecting a molecule of interest in a biological samplealso is disclosed. One embodiment of the method comprised contacting thebiological sample with a hapten-antibody conjugate comprising anantibody linked to the hapten using a heterobifunctional PEG linker, thehapten being selected from oxazoles, pyrazoles, thiazoles, nitroaryls,benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarins,podophyllotoxin-based compounds, and combinations thereof. A signalgenerated by the hapten-antibody conjugate is detected after treatmentwith an anti-hapten antibody having at least one detectable label.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic drawing illustrating one embodiment of amultiplexed in situ hybridization process.

FIG. 2 is a schematic drawing illustrating one embodiment of a methodfor using enzymes as signal generating moieties.

FIG. 3 is a schematic drawing illustrating direct detection of a targetusing a primary antibody comprising a detectable signal generatinglabel.

FIG. 4 is a schematic diagram illustrating one embodiment of a methodfor amplifying detection signals.

FIG. 5 is a schematic drawing illustrating one embodiment ofhapten-quantum dot immunohistochemistry detection according to thepresent invention.

FIG. 6 is a photomicrograph depicting IHC positive staining ofanti-hapten antibody detection using a primary antibody conjugated to adisclosed embodiment of a class of haptens according to the presentinvention.

FIG. 7 is a photomicrograph depicting IHC negative staining using ananti-hapten antibody using a primary antibody conjugated to a disclosedembodiment of a class of haptens according to the present invention.

FIG. 8 is a schematic diagram illustrating one embodiment of a disclosedmethod for multiplexed detection of multiple targets in a sample usingplural different haptens and plural different signal generating moietiesto generate plural different detectable signals.

FIG. 9 is a photomicrograph depicting using multiple haptens andantibodies thereto, such as antibiotin and antidinitrophenyl, fordetection of sample, such as a protein, in tissue.

FIG. 10 schematically illustrates one embodiment of multiplexeddetection of two different classes of targets, namely gene and proteintargets.

FIG. 11 is a photomicrograph depicting detection of protein and 2 genes,such as by using an antidinitrophenyl antibody.

FIG. 12 illustrates hybridoma fusion product ELISA results for mouse IgGmonoclonal antibodies against one embodiment of a disclosed benzofurazanhapten.

FIG. 13 illustrates hybridoma fusion product ELISA results for mouse IgGmonoclonal antibodies against one embodiment of a disclosed benzofurazanhapten.

FIG. 14 illustrates hybridoma fusion product ELISA results for mouse IgGmonoclonal antibodies against one embodiment of a disclosed benzofurazanhapten.

FIG. 15 illustrates hybridoma fusion product ELISA results for mouse IgGmonoclonal antibodies against one embodiment of a disclosed benzofurazanhapten.

FIG. 16 illustrates hybridoma fusion product ELISA results for mouse IgGmonoclonal antibodies against one embodiment of a disclosed benzofurazanhapten.

FIG. 17 is a graph of percent nucleotide versus DNP concentration.

FIG. 18 is a graph of salmon DNA concentration on percent nucleotidelabeled.

FIG. 19 is a chromogenic IHC staining of CD20-biotin-labeled primaryantibodies with anti-biotin HRP conjugates on normal tonsil tissue.

FIG. 20 is a chromogenic IHC staining of a CD45 thiazolesulfonamide-based hapten-labeled primary antibodies with anti-thiazolesulfonamide HRP conjugates on normal tonsil tissue.

FIG. 21 is a chromogenic IHC staining of a Ki-67 benzofurazan-basedhapten-labeled primary antibodies with anti-benzofurazan HRP conjugateson normal tonsil tissue.

FIG. 22 is a chromogenic IHC staining of a CD34 nitropyrazole-basedhapten-labeled primary antibodies with anti-nitropyrazole HRP conjugateson normal tonsil tissue.

FIG. 23 is a chromogenic IHC staining of a Kappa dinitrophenyl-basedhapten-labeled primary antibodies with anti-dinitrophenyl HRP conjugateson normal tonsil tissue.

FIG. 24 is a chromogenic IHC staining of Lambda rotenone-basedhapten-labeled primary antibodies with anti-rotenone HRP conjugates onnormal tonsil tissue.

FIG. 25 is a fluorescent IHC staining in normal tonsil using anti-lambdaattached to biotin and detected with anti-biotin antibody QDotconjugate.

FIG. 26 is a fluorescent IHC staining in normal tonsil usingantil-lambda attached to thiazole sulfonamide-based hapten and detectedwith anti-thiazole sulfonamide antibody QDot conjugate.

FIG. 27 is a fluorescent IHC staining in normal tonsil usingantil-lambda attached to benzofurazan-based hapten and detected withanti-benzofurazan antibody QDot conjugate.

FIG. 28 is a fluorescent IHC staining in normal tonsil usingantil-lambda attached to dinitrophenyl-based hapten and detected withanti-dinitrophenyl antibody QDot conjugate.

FIG. 29 is a fluorescent IHC staining in normal tonsil usingantil-lambda attached to nitropyrazole-based hapten and detected withanti-nitropyrazole antibody QDot conjugate.

FIG. 30 is a fluorescent IHC staining in normal tonsil usingantil-lambda attached to rotenone-based hapten and detected withanti-rotenone antibody QDot conjugate.

FIG. 31 is a fluorescent IHC staining of biotin labeled CD20 primaryantibody and anti-biotin QDot 525 conjugate on normal tonsil tissue.

FIG. 32 is a fluorescent IHC staining of thiazole sulfonamide-basedhapten labeled CD45 primary antibody and anti-thiazole sulfonamideantibody QDot 565 conjugate on normal tonsil tissue.

FIG. 33 is a fluorescent IHC staining of benzofurazan-based haptenlabeled Ki67 primary antibody and anti-benzofurazan antibody QDot 585conjugate on normal tonsil tissue.

FIG. 34 is a fluorescent IHC staining of dinitrophenyl-based haptenlabeled Kappa primary antibody and anti-dinitrophenyl antibody QDot 605conjugate on normal tonsil tissue.

FIG. 35 is a fluorescent IHC staining of nitropyrazole-based haptenlabeled CD34 primary antibodies anti-nitropyrazole antibody QDot 655conjugate on normal tonsil tissue.

FIG. 36 is a fluorescent IHC staining of rotenone-based hapten labeledLambda primary antibodies and anti-rotenone antibody QDot 705 conjugateon normal tonsil tissue.

FIG. 37 is multiplexed staining composite using a mixture of the primaryantibody-hapten conjugates and sequentially visualized with a mixture ofanti-hapten antibody QDot conjugates on normal tonsil tissue as statedin FIGS. 31-36 and in Example 34.

FIG. 38 is an image extracted from a multiplexed staining composite thatrepresents anti CD45-thiazole sulfonamide-conjugate and hapten primaryantibody and anti-thiazole sulfonamide antibody QDot 565 conjugate onnormal tonsil tissue.

FIG. 39 is an image extracted from a multiplexed staining composite fromFIG. 37 that represents the staining from the Ki67benzofurazan-conjugate and hapten primary antibody andanti-benzofurazan-based antibody QDot 585 conjugate on normal tonsiltissue.

FIG. 40 is an image extracted from a multiplexed staining composite fromFIG. 37 that represents anti Kappa-dinitrophenyl-conjugate and haptenprimary antibody and anti-dinitrophenyl-based hapten antibody QDot 605conjugates on normal tonsil tissue.

FIG. 41 is an image extracted from a multiplexed staining composite fromFIG. 37 with anti CD34 nitropyrazole-conjugate and hapten primaryantibody and anti-nitropyrazole-based hapten antibody QDot 655 conjugateon normal tonsil tissue.

FIG. 42 is an image extracted from a multiplexed staining composite fromFIG. 37 with anti CD20 biotin primary antibody QDot 525 conjugate onnormal tonsil tissue.

FIG. 43 is an image extracted from a multiplexed staining composite fromFIG. 37 with Lambda-rotenone-conjugate and hapten primary antibody andanti-rotenone-based hapten antibody QDot 705 conjugate on normal tonsiltissue.

FIG. 44 is a graph of wavelength versus relative fluorescence thatrepresents the wavelengths used to extract individual QDot signals fromthe multiplexed staining composite of FIG. 37 and shows the signal isabove the nominal autofluorescence of the tonsil tissue.

DETAILED DESCRIPTION I. Terms and Introduction

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all nucleotide sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides or other compounds are approximate, andare provided for description. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present disclosure, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingexplanations of terms, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

In order to facilitate review of the various examples of thisdisclosure, the following explanations of specific terms are provided:

Adjuvant: A substance that non-specifically enhances the immune responseto an antigen. Development of vaccine adjuvants for use in humans isreviewed in Singh et al. (Nat. Biotechnol. 17:1075-1081, 1999), whichdiscloses that, at the time of its publication, aluminum salts, such asaluminum hydroxide (Amphogel, Wyeth Laboratories, Madison, N.J.), andthe MF59 microemulsion are the only vaccine adjuvants approved for humanuse. An aluminum hydrogel (available from Brentg Biosector, Copenhagen,Denmark is another common adjuvant).

In one embodiment, an adjuvant includes a DNA motif that stimulatesimmune activation, for example the innate immune response or theadaptive immune response by T-cells, B-cells, monocytes, dendriticcells, and natural killer cells. Specific, non-limiting examples of aDNA motif that stimulates immune activation include CpGoligodeoxynucleotides, as described in U.S. Pat. Nos. 6,194,388;6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and6,429,199.

Amplification: Certain embodiments of the present invention allow asingle target to be detected using plural visualization complexes, wherethe complexes can be the same or different, to facilitate identificationand/or quantification of a particular target.

Analog, Derivative or Mimetic: An analog is a molecule that differs inchemical structure from a parent compound, for example a homolog(differing by an increment in the chemical structure, such as adifference in the length of an alkyl chain), a molecular fragment, astructure that differs by one or more functional groups, a change inionization. Structural analogs are often found using quantitativestructure activity relationships (QSAR), with techniques such as thosedisclosed in Remington (The Science and Practice of Pharmacology, 19thEdition (1995), chapter 28). A derivative is a biologically activemolecule derived from the base structure. A mimetic is a molecule thatmimics the activity of another molecule, such as a biologically activemolecule. Biologically active molecules can include chemical structuresthat mimic the biological activities of a compound.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, and cows.

Antibody: “Antibody” collectively refers to immunoglobulins orimmunoglobulin-like molecules (including by way of example and withoutlimitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, andsimilar molecules produced during an immune response in any vertebrate,for example, in mammals such as humans, goats, rabbits and mice) andantibody fragments that specifically bind to a molecule of interest (ora group of highly similar molecules of interest) to the substantialexclusion of binding to other molecules (for example, antibodies andantibody fragments that have a binding constant for the molecule ofinterest that is at least 10³ M⁻¹ greater, at least 10⁴ M⁻¹ greater orat least 10⁵ M⁻¹ greater than a binding constant for other molecules ina biological sample.

More particularly, “antibody” refers to a polypeptide ligand comprisingat least a light chain or heavy chain immunoglobulin variable regionwhich specifically recognizes and binds an epitope of an antigen.Antibodies are composed of a heavy and a light chain, each of which hasa variable region, termed the variable heavy (V_(H)) region and thevariable light (V_(L)) region. Together, the V_(H) region and the V_(L)region are responsible for binding the antigen recognized by theantibody.

This includes intact immunoglobulins and the variants and portions ofthem well known in the art. Antibody fragments include proteolyticantibody fragments [such as F(ab′)₂ fragments, Fab′ fragments, Fab′-SHfragments and Fab fragments as are known in the art], recombinantantibody fragments (such as sFv fragments, dsFv fragments, bispecificsFv fragments, bispecific dsFv fragments, F(ab)′₂ fragments, singlechain Fv proteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”),diabodies, and triabodies (as are known in the art), and camelidantibodies (see, for example, U.S. Pat. Nos. 6,015,695;6,005,079-5,874,541; 5,840,526; 5,800,988; and 5,759,808). A scFvprotein is a fusion protein in which a light chain variable region of animmunoglobulin and a heavy chain variable region of an immunoglobulinare bound by a linker, while in dsFvs, the chains have been mutated tointroduce a disulfide bond to stabilize the association of the chains.The term also includes genetically engineered forms such as chimericantibodies (for example, humanized murine antibodies), heteroconjugateantibodies (such as, bispecific antibodies). See also, Pierce Catalogand Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs”. The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The Kabat database is now maintained online. The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDRs inthree-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds RET will have a specificV_(H) region and the V_(L) region sequence, and thus specific CDRsequences. Antibodies with different specificities (i.e. differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

Antigen: A compound, composition, or substance that may be specificallybound by the products of specific humoral or cellular immunity, such asan antibody molecule or T-cell receptor. Antigens can be any type ofmolecule including, for example, haptens, simple intermediarymetabolites, sugars (e.g., oligosaccharides), lipids, and hormones aswell as macromolecules such as complex carbohydrates (e.g.,polysaccharides), phospholipids, nucleic acids and proteins. Commoncategories of antigens include, but are not limited to, viral antigens,bacterial antigens, fungal antigens, protozoa and other parasiticantigens, tumor antigens, antigens involved in autoimmune disease,allergy and graft rejection, toxins, and other miscellaneous antigens.In one example, an antigen is a Bacillus antigen, such as γPGA.

Avidin: Any type of protein that specifically binds biotin to thesubstantial exclusion of other small molecules that might be present ina biological sample. Examples of avidin include avidins that arenaturally present in egg white, oilseed protein (e.g., soybean meal),and grain (e.g., corn/maize) and streptavidin, which is a protein ofbacterial origin.

Binding affinity: The tendency of one molecule to bind (typicallynon-covalently) with another molecule, such as the tendency of a memberof a specific binding pair for another member of a specific bindingpair. A binding affinity can be measured as a binding constant, whichbinding affinity for a specific binding pair (such as anantibody/antigen pair or nucleic acid probe/nucleic acid sequence pair)can be at least 1×10⁵ M⁻¹, such as at least 1×10⁶ M⁻¹, at least 1×10⁷M⁻¹ or at least 1×108 M⁻¹. In one embodiment, binding affinity iscalculated by a modification of the Scatchard method described byFrankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment,binding affinity is measured by an antigen/antibody dissociation rate.In yet another embodiment, a high binding affinity is measured by acompetition radioimmunoassay. In several examples, a high bindingaffinity for an antibody/antigen pair is at least about 1×10⁸ M⁻¹. Inother embodiments, a high binding affinity is at least about 1.5×10⁸M⁻¹, at least about 2.0×10⁸ M⁻¹, at least about 2.5×10⁸ M⁻¹, at leastabout 3.0×10⁸ M⁻¹, at least about 3.5×10⁸ M⁻¹, at least about 4.0×10⁸M⁻¹, at least about 4.5×10⁸ M⁻¹, or at least about 5.0×10⁸ M⁻¹.

Carrier: A molecule to which a hapten or an antigen can be bound.Carrier molecules include immunogenic carriers and specific-bindingcarriers. When bound to an immunogenic carrier, the bound molecule maybecome immunogenic. Immunogenic carriers may be chosen to increase theimmunogenicity of the bound molecule and/or to elicit antibodies againstthe carrier, which are diagnostically, analytically, and/ortherapeutically beneficial. Covalent linking of a molecule to a carriercan confer enhanced immunogenicity and T-cell dependence (Pozsgay etal., PNAS 96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976;Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers includepolymeric carriers, which can be natural (for example, proteins frombacteria or viruses), semi-synthetic or synthetic materials containingone or more functional groups to which a reactant moiety can beattached. Specific binding carriers can by any type of specific bindingmoiety, including an antibody, a nucleic acid, an avidin, aprotein-nucleic acid.

Examples of suitable immunogenic carriers are those that can increasethe immunogenicity of a hapten and/or help elicit antibodies against thehapten which are diagnostically, analytically, and/or therapeuticallybeneficial. Useful carriers include polymeric carriers, which can benatural (such as proteins like ovalbumin or keyhole limpet hemocyanin)or derived from a natural polymer isolated from any organism (includingviruses), semi-synthetic or synthetic materials containing one or morefunctional groups, for example primary and/or secondary amino groups,azido groups, hydroxyl groups, or carboxyl groups, to which a reactantmoiety can be attached. The carrier can be water soluble or insoluble,and in some embodiments is a protein or polypeptide. Carriers thatfulfill these criteria are generally known in the art (see, for example,Fattom et al., Infect. Immun. 58:2309-12, 1990; Devi et al., PNAS88:7175-79, 1991; Szu et al., Infect. Immun. 59:4555-61, 1991; Szu etal., J. Exp. Med. 166:1510-24, 1987; and Pavliakova et al., Infect.Immun. 68:2161-66, 2000).

The immunogenic carrier can be a polypeptide, such as a polypeptide of arotavirus, or of a virus other than a rotavirus. A non limiting, and farfrom exhaustive list of such other viruses includes Adeno-associatedvirus, Adenovirus, Avian infectious bronchitis virus, Baculovirus,Chicken pox, Corona virus, Cytomegalovirus, Distemper, Enterovirus,Epstein Barr virus, Feline leukemia virus, Flavivirus, Foot and mouthdisease virus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E,Herpes species, Herpes simplex, Influenza virus, HIV-1, HIV-2, HTLV 1,Influenza A and B, Kunjin virus, Lassa fever virus, LCMV (lymphocyticchoriomeningitis virus), lentivirus, Measles, Mengo virus,Morbillivirus, Myxovirus, Papilloma virus, Parovirus, Parainfluenzavirus, Paramyxovirus, Parvovirus, Poko virus, Polio virus, Polyomatumour virus, pseudorabies, Rabies virus, Reovirus, Respiratorysyncytial virus, retrovirus, rhinovirus, Rinderpest, Rotavirus, Semlikiforest virus, Sendai virus, Simian Virus 40, Sindbis virus, SV5, Tickborne encephalitis virus, Togavirus (rubella, yellow fever, denguefever), Vaccinia virus, Venezuelan equine encephalomyelitis, Vesicularstomatis virus, metapneumovirus, norovirus, SARS virus, smallpox virus,picornaviruses, varicella zoster, and West Nile virus.

Alternatively, the immunogenic carrier polypeptide can be that of abacteria or other pathogenic organism. Exemplary bacterial polypeptidesinclude those of Achromobacter xylosoxidans, Acinetobactercalcoaceticus, preferably A. anitratus, A. haemolyticus, A. alcaligenes,and A. lwoffii, Actinomyces israelii, Aeromonas hydrophilia, Alcaligenesspecies, preferably A. faecalis, A. odorans and A. denitrificans,Arizona hinshawii, Bacillus anthracis, Bacillus cereus, Bacteroidesfragilis, Bacteroides melaninogenicus, Bordetella pertussis, Borreliaburgdorferi, Borrelia recurrentis, Brucella species, preferably B.abortus, B. suis, B. melitensis and B. canis, Calymmatobacteriumgranulomatis, Campylobacter coli (e.g., the CjaA polypeptide),Campylobacter fetus ssp. intestinalis, Campylobacter fetus ssp. jejuni,Chlamydia species, preferably C. psittaci and C. trachomatis,Chromobacterium violaceum, Citrobacter species, preferably C. freundiiand C. diversus, Clostridium botulinum, Clostridium perfringens,Clostridium difficile, Clostridium tetani, Corynebacterium diphtheriae,Corynebacterium, preferably C. ulcerans, C. haemolyticum and C.pseudotuberculosis, Coxiella burnetii, Edwardsiella tarda, Eikenellacorrodens, Enterobacter, preferably E. cloacae, E. aerogenes, E. hafniae(also named Hafnia alvei) and E. agglomerans, Erysipelothrixrhusiopathiae, Escherichia coli, Flavobacterium meningosepticum,Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter species (e.g.,the UreB polypeptide of H. pylori), Klebsiella species, preferably K.pneumoniae, K. ozaenae og K. rhinoscleromatis, Legionella species,Leptospira interrogans, Listeria monocytogenes, Moraxella species,preferably M. lacunata and M. osloensis, Mycobacterium bovis,Mycobacterium leprae, Mycobacterium tuberculosis (e.g., the CFP10polypeptide), Mycoplasma species, preferably M. pneumoniae, Neisseriagonorrhoeae, Neisseria meningitidis, Nocardia species, preferably N.asteroides and N. brasiliensis, Pasteurella haemolytica, Pasteurellamultocida, Peptococcus magnus, Plesiomonas shigelloides, Pneumococci,Proteus species, preferably P. mirabilis, P. vulgaris, P. rettgeri andP. morganii (also named Providencia rettgeri and Morganella morganiirespectively), Providencia species, preferably P. alcalifaciens, P.stuartii and P. rettgeri (also named Proteus rettgeri), Pseudomonasaeruginosa, Pseudomonas mallei, Pseudomonas pseudomallei, Rickettsia,Rochalimaia henselae, Salmonella species, preferably S. enteridis, S.typhi and S. derby, and most preferably Salmonella species of the typeSalmonella DT104, Serratia species, preferably S. marcescens, Shigelladysenteriae, S. flexneri, S. boydii and S. sonnei, Spirillum minor,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptobacillus moniliformis, Streptococcus, preferablyS. faecalis, S. faecium and S. durans, Streptococcus agalactiae,Streptococcus pneumoniae, Streptococcus pyogenes (e.g., the Sfb1polypeptide), Treponema carateum, Treponema pallidum, Treponemapertenue, preferably T. pallidum, Ureaplasma urealyticum, Vibriocholerae, Vibrio parahaemolyticus, Yersinia enterocolitica, and Yersiniapestis.

Parasitic immunogenic carriers may for example be isolated and/orderived from Malaria (Plasmodium falciparum, P. vivax, P. malariae),Schistosomes, Trypanosomes, Leishmania, Filarial nematodes,Trichomoniasis, Sarcosporidiasis, Taenia (T. saginata, T. solium),Leishmania, Toxoplasma gondii, Trichinelosis (Trichinella spiralis) orCoccidiosis (Eimeria species).

Illustrative fungal immunogenic carriers can be isolated and/or derivedfrom a fungus selected from Cryptococcus neoformans, Candida albicans,Aspergillus fumigatus and Coccidioidomycosis.

Specific, non-limiting examples of water soluble polypeptide immunogeniccarriers include, but are not limited to, natural, semi-synthetic orsynthetic polypeptides or proteins from bacteria or viruses. In oneembodiment, bacterial products for use as carriers include bacterialwall proteins and other products (for example, streptococcal orstaphylococcal cell walls), and soluble antigens of bacteria. In anotherembodiment, bacterial products for use as carriers include bacterialtoxins. Bacterial toxins include bacterial products that mediate toxiceffects, inflammatory responses, stress, shock, chronic sequelae, ormortality in a susceptible host.

Specific, non-limiting examples of water insoluble polymeric carriersinclude, but are not limited to, aminoalkyl agarose (for example,aminopropyl or aminohexyl SEPHAROSE; Pharmacia Inc., Piscataway, N.J.),aminopropyl glass, cross-linked dextran, and the like, to which areactive moiety can be attached. Other carriers can be used, providedthat a functional group is available for covalently attaching a reactivegroup.

Chimeric antibody: An antibody that has framework residues from onespecies, such as human, and CDRs (which generally confer antigenbinding) from another species, such as a murine antibody thatspecifically binds RET.

Conjugating, joining, bonding or linking: Covalently linking onemolecule to another molecule to make a larger molecule. For example,making two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a hapten or other molecule to a polypeptide, suchas an scFv antibody. In the specific context, the terms includereference to joining a ligand, such as an antibody moiety, to aneffector molecule (“EM”). The linkage can be either by chemical orrecombinant means. “Chemical means” refers to a reaction between theantibody moiety and the effector molecule such that there is a covalentbond formed between the two molecules to form one molecule.

Coupled: When applied to a first atom or molecule being “coupled” to asecond atom or molecule can be both directly coupled and indirectlycoupled. A secondary antibody provides an example of indirect coupling.One specific example of indirect coupling is a rabbit anti-haptenprimary antibody that is bound by a mouse anti-rabbit IgG antibody, thatis in turn bound by a goat anti-mouse IgG antibody that is covalentlylinked to a detectable label.

Epitope: An antigenic determinant. These are particular chemical groupsor contiguous or non-contiguous peptide sequences on a molecule that areantigenic, that is, that elicit a specific immune response. An antibodybinds a particular antigenic epitope.

Hapten: A molecule, typically a small molecule that can combinespecifically with an antibody, but typically is substantially incapableof being immunogenic except in combination with a carrier molecule.

Homopolymer: This term refers to a polymer formed by the bondingtogether of multiple units of a single type of molecular species, suchas a single monomer (for example, an amino acid).

Humanized antibody: An antibody comprising a humanized light chain and ahumanized heavy chain immunoglobulin. A humanized antibody binds to thesame antigen as the donor antibody that provides the CDRs. The acceptorframework of a humanized immunoglobulin or antibody may have a limitednumber of substitutions by amino acids taken from the donor framework.Humanized or other monoclonal antibodies can have additionalconservative amino acid substitutions which have substantially no effecton antigen binding or other immunoglobulin functions. Humanizedimmunoglobulins can be constructed by means of genetic engineering (seefor example, U.S. Pat. No. 5,585,089).

Humanized immunoglobulin: an immunoglobulin including a human frameworkregion and one or more CDRs from a non-human (for example a mouse, rat,or synthetic) immunoglobulin. The non-human immunoglobulin providing theCDRs is termed a “donor,” and the human immunoglobulin providing theframework is termed an “acceptor.” In one embodiment, all the CDRs arefrom the donor immunoglobulin in a humanized immunoglobulin. Constantregions need not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences.

Immune Response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus. Animmune response can include any cell of the body involved in a hostdefense response for example, an epithelial cell that secretesinterferon or a cytokine. An immune response includes, but is notlimited to, an innate immune response or inflammation.

Immunogenic Conjugate or Composition: A term used herein to mean acomposition useful for stimulating or eliciting a specific immuneresponse (or immunogenic response) in a vertebrate. In some embodiments,the immunogenic response is protective or provides protective immunity,in that it enables the vertebrate animal to better resist infection ordisease progression from the organism against which the immunogeniccomposition is directed. One specific example of a type of immunogeniccomposition is a vaccine.

Immunogen: A compound, composition, or substance which is capable, underappropriate conditions, of stimulating the production of antibodies or aT-cell response in an animal, including compositions that are injectedor absorbed into an animal.

Immunologically Effective Dose: An immunologically effective dose of thedisclosed conjugates of the disclosure is therapeutically effective andwill prevent, treat, lessen, or attenuate the severity, extent orduration of a disease or condition.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (such as temperature, osmolarity, pH) that are typical insidea living mammal or a mammalian cell. While it is recognized that someorgans are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (i.e., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Inhibiting or Treating a Disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as anthrax. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop. As used herein,the term “ameliorating,” with reference to a disease, pathologicalcondition or symptom, refers to any observable beneficial effect of thetreatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease, a slower progression of the disease, a reduction in the numberof relapses of the disease, an improvement in the overall health orwell-being of the subject, or by other parameters well known in the artthat are specific to the particular disease.

Isolated: An “isolated” microorganism (such as a virus, bacterium,fungus, or protozoan) has been substantially separated or purified awayfrom microorganisms of different types, strains, or species.Microorganisms can be isolated by a variety of techniques, includingserial dilution and culturing.

An “isolated” biological component (such as a nucleic acid molecule,protein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, such as other chromosomal andextra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell, as well as chemically synthesized nucleic acids orproteins, or fragments thereof.

Detectable Label: A detectable compound or composition that is attacheddirectly or indirectly to another molecule, such as an antibody or aprotein, to facilitate detection of that molecule. Specific,non-limiting examples of labels include fluorescent tags, enzymes, andradioactive isotopes.

Linker peptide: A peptide within an antibody binding fragment (such asan Fv fragment) which serves to indirectly bond the variable heavy chainto the variable light chain. “Linker” can also refer to a peptideserving to link a targeting moiety, such as a scFv, to an effectormolecule, such as a cytotoxin or a detectable label.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Molecule of interest or Target: A molecule for which the presence,location and/or concentration is to be determined. Examples of moleculesof interest include proteins and nucleic acid sequences tagged withhaptens.

Monoclonal antibody: An antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

Multiplex, -ed, -ing: Embodiments of the present invention allowmultiple targets in a sample to be detected substantiallysimultaneously, or sequentially, as desired, using plural differentconjugates. Multiplexing can include identifying and/or quantifyingnucleic acids generally, DNA, RNA, peptides, proteins, both individuallyand in any and all combinations. Multiplexing also can include detectingtwo or more of a gene, a messenger and a protein in a cell in itsanatomic context.

Nanoparticle: A nanoscale particle with a size that is measured innanometers, for example, a nanoscopic particle that has at least onedimension of less than about 100 nm. Examples of nanoparticles includeparamagnetic nanoparticles, superparamagnetic nanoparticles, metalnanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers(such as with covalently attached metal chelates), nanofibers,nanohorns, nano-onions, nanorods, nanoropes and quantum dots. Ananoparticle can produce a detectable signal, for example, throughabsorption and/or emission of photons (including radio frequency andvisible photons) and plasmon resonance.

Neoplasia and Tumor: The process of abnormal and uncontrolled growth ofcells. Neoplasia is one example of a proliferative disorder.

The product of neoplasia is a neoplasm (a tumor), which is an abnormalgrowth of tissue that results from excessive cell division. A tumor thatdoes not metastasize is referred to as “benign.” A tumor that invadesthe surrounding tissue and/or can metastasize is referred to as“malignant.” Examples of hematological tumors include leukemias,including acute leukemias (such as acute lymphocytic leukemia, acutemyelocytic leukemia, acute myelogenous leukemia and myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronicleukemias (such as chronic myelocytic (granulocytic) leukemia, chronicmyelogenous leukemia, and chronic lymphocytic leukemia), polycythemiavera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent andhigh grade forms), multiple myeloma, Waldenstrom's macroglobulinemia,heavy chain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors(such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds or molecules, such as one or more SARS-CoV nucleicacid molecules, proteins or antibodies that bind these proteins, andadditional pharmaceutical agents. The term “pharmaceutically acceptablecarrier” should be distinguished from “carrier” as described above inconnection with a hapten/carrier conjugate or an antigen/carrierconjugate.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced.

The term “residue” or “amino acid residue” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.

Protein: A molecule, particularly a polypeptide, comprised of aminoacids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpeptide, protein, conjugate, or other active compound is one that isisolated in whole or in part from proteins or other contaminants.Generally, substantially purified peptides, proteins, conjugates, orother active compounds for use within the disclosure comprise more than80% of all macromolecular species present in a preparation prior toadmixture or formulation of the peptide, protein, conjugate or otheractive compound with a pharmaceutical carrier, excipient, buffer,absorption enhancing agent, stabilizer, preservative, adjuvant or otherco-ingredient in a complete pharmaceutical formulation for therapeuticadministration. More typically, the peptide, protein, conjugate or otheractive compound is purified to represent greater than 90%, often greaterthan 95% of all macromolecular species present in a purified preparationprior to admixture with other formulation ingredients. In other cases,the purified preparation may be essentially homogeneous, wherein othermacromolecular species are not detectable by conventional techniques.

Quantum dot: A nanoscale particle that exhibits size-dependentelectronic and optical properties due to quantum confinement. Quantumdots have, for example, been constructed of semiconductor materials(e.g., cadmium selenide and lead sulfide) and from crystallites (grownvia molecular beam epitaxy), etc. A variety of quantum dots havingvarious surface chemistries and fluorescence characteristics arecommercially available from Invitrogen Corporation, Eugene, Oreg. (see,for example, U.S. Pat. Nos. 6,815,064, 6,682,596 and 6,649,138, each ofwhich patents is incorporated by reference herein). Quantum dots arealso commercially available from Evident Technologies (Troy, N.Y.).Other quantum dots include alloy quantum dots such as ZnSSe, ZnSeTe,ZnSTe, CdSSe, CdSeTe, ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe,ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe,ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, and InGaN quantumdots (Alloy quantum dots and methods for making the same are disclosed,for example, in US Application Publication No. 2005/0012182 and PCTPublication WO 2005/001889).

Reactive Groups: Formulas throughout this application refer to “reactivegroups,” which can be any of a variety of groups suitable for coupling afirst unit to a second unit as described herein. For example, thereactive group might be an amine-reactive group, such as anisothiocyanate, an isocyanate, an acyl azide, an NHS ester, an acidchloride, such as sulfonyl chloride, aldehydes and glyoxals, epoxidesand oxiranes, carbonates, arylating agents, imidoesters, carbodiimides,anhydrides, and combinations thereof. Suitable thiol-reactive functionalgroups include haloacetyl and alkyl halides, maleimides, aziridines,acryloyl derivatives, arylating agents, thiol-disulfide exchangereagents, such as pyridyl disulfides, TNB-thiol, and disulfidereductants, and combinations thereof. Suitable carboxylate-reactivefunctional groups include diazoalkanes, diazoacetyl compounds,carbonyldiimidazole compounds, and carbodiimides. Suitablehydroxyl-reactive functional groups include epoxides and oxiranes,carbonyldiimidazole, N,N′-disuccinimidyl carbonates orN-hydroxysuccinimidyl chloroformates, periodate oxidizing compounds,enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde andketone-reactive functional groups include hydrazines, Schiff bases,reductive amination products, Mannich condensation products, andcombinations thereof. Active hydrogen-reactive compounds includediazonium derivatives, mannich condensation products, iodinationreaction products, and combinations thereof. Photoreactive chemicalfunctional groups include aryl azides, halogenated aryl azides,benzophonones, diazo compounds, diazirine derivatives, and combinationsthereof.

Sample: A biological specimen containing genomic DNA, RNA (includingmRNA), protein, or combinations thereof, obtained from a subject.Examples include, but are not limited to, peripheral blood, urine,saliva, tissue biopsy, surgical specimen, amniocentesis samples andautopsy material. In one example, a sample includes a biopsy of anadenocarcinoma, a sample of noncancerous tissue, a sample of normaltissue (from a subject not afflicted with a known disease or disorder).

Specific binding moiety: A member of a specific-binding pair. Specificbinding pairs are pairs of molecules that are characterized in that theybind each other to the substantial exclusion of binding to othermolecules (for example, specific binding pairs can have a bindingconstant that is at least 10³ M⁻¹ greater, 10⁴ M⁻¹ greater or 10⁵ M⁻¹greater than a binding constant for either of the two members of thebinding pair with other molecules in a biological sample). Particularexamples of specific binding moieties include specific binding proteins(for example, antibodies, lectins, avidins such as streptavidins, andprotein A), nucleic acids sequences, and protein-nucleic acids. Specificbinding moieties can also include the molecules (or portions thereof)that are specifically bound by such specific binding proteins.

Therapeutically Effective Amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of a conjugate useful inincreasing resistance to, preventing, ameliorating, and/or treatinginfection and disease. Ideally, a therapeutically effective amount of anagent is an amount sufficient to increase resistance to, prevent,ameliorate, and/or treat infection and without causing a substantialcytotoxic effect in the subject. The effective amount of an agent usefulfor increasing resistance to, preventing, ameliorating, and/or treatinginfection and disease in a subject will be dependent on the subjectbeing treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition.

Vaccine: A vaccine is a pharmaceutical composition that elicits aprophylactic or therapeutic immune response in a subject. In some cases,the immune response is a protective response. Typically, a vaccineelicits an antigen-specific immune response to an antigen of a pathogen,for example, a bacterial or viral pathogen, or to a cellular constituentcorrelated with a pathological condition. A vaccine may include apolynucleotide, a peptide or polypeptide, a polysaccharide, a virus, abacteria, a cell or one or more cellular constituents. In some cases,the virus, bacteria or cell may be inactivated or attenuated to preventor reduce the likelihood of infection, while maintaining theimmunogenicity of the vaccine constituent.

The antigenic polypeptide can be that of a rotavirus, or of a virusother than a rotavirus. A non limiting, and far from exhaustive list ofsuch other viruses includes Adeno-associated virus, Adenovirus, Avianinfectious bronchitis virus, Baculovirus, Chicken pox, Corona virus,Cytomegalovirus, Distemper, Enterovirus, Epstein Barr virus, Felineleukemia virus, Flavivirus, Foot and mouth disease virus, Hepatitis A,Hepatitis B, Hepatitis C, Hepatitis E, Herpes species, Herpes simplex,Influenza virus, HIV-1, HIV-2, HTLV 1, Influenza A and B, Kunjin virus,Lassa fever virus, LCMV (lymphocytic choriomeningitis virus),lentivirus, Measles, Mengo virus, Morbillivirus, Myxovirus, Papillomavirus, Parovirus, Parainfluenza virus, Paramyxovirus, Parvovirus, Pokovirus, Polio virus, Polyoma tumour virus, pseudorabies, Rabies virus,Reovirus, Respiratory syncytial virus, retrovirus, rhinovirus,Rinderpest, Rotavirus, Semliki forest virus, Sendai virus, Simian Virus40, Sindbis virus, SV5, Tick borne encephalitis virus, Togavirus(rubella, yellow fever, dengue fever), Vaccinia virus, Venezuelan equineencephalomyelitis, Vesicular stomatis virus, metapneumovirus, norovirus,SARS virus, smallpox virus, picornaviruses, varicella zoster, and WestNile virus.

Alternatively, the antigenic polypeptide can be that of a bacteria orother pathogenic organism. Exemplary bacterial polypeptides includethose of Achromobacter xylosoxidans, Acinetobacter calcoaceticus,preferably A. anitratus, A. haemolyticus, A. alcaligenes, and A.lwoffii, Actinomyces israelii, Aeromonas hydrophilia, Alcaligenesspecies, preferably A. faecalis, A. odorans and A. denitrificans,Arizona hinshawii, Bacillus anthracis, Bacillus cereus, Bacteroidesfragilis, Bacteroides melaninogenicus, Bordetella pertussis, Borreliaburgdorferi, Borrelia recurrentis, Brucella species, preferably B.abortus, B. suis, B. melitensis and B. canis, Calymmatobacteriumgranulomatis, Campylobacter coli (e.g., the CjaA polypeptide),Campylobacter fetus ssp. intestinalis, Campylobacter fetus ssp. jejuni,Chlamydia species, preferably C. psittaci and C. trachomatis,Chromobacterium violaceum, Citrobacter species, preferably C. freundiiand C. diversus, Clostridium botulinum, Clostridium perfringens,Clostridium difficile, Clostridium tetani, Corynebacterium diphtheriae,Corynebacterium, preferably C. ulcerans, C. haemolyticum and C.pseudotuberculosis, Coxiella burnetii, Edwardsiella tarda, Eikenellacorrodens, Enterobacter, preferably E. cloacae, E. aerogenes, E. hafniae(also named Hafnia alvei) and E. agglomerans, Erysipelothrixrhusiopathiae, Escherichia coli, Flavobacterium meningosepticum,Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter species (e.g.,the UreB polypeptide of H. pylori), Klebsiella species, preferably K.pneumoniae, K. ozaenae og K. rhinoscleromatis, Legionella species,Leptospira interrogans, Listeria monocytogenes, Moraxella species,preferably M. lacunata and M. osloensis, Mycobacterium bovis,Mycobacterium leprae, Mycobacterium tuberculosis (e.g., the CFP10polypeptide), Mycoplasma species, preferably M. pneumoniae, Neisseriagonorrhoeae, Neisseria meningitidis, Nocardia species, preferably N.asteroides and N. brasiliensis, Pasteurella haemolytica, Pasteurellamultocida, Peptococcus magnus, Plesiomonas shigelloides, Pneumococci,Proteus species, preferably P. mirabilis, P. vulgaris, P. rettgeri andP. morganii (also named Providencia rettgeri and Morganella morganiirespectively), Providencia species, preferably P. alcalifaciens, P.stuartii and P. rettgeri (also named Proteus rettgeri), Pseudomonasaeruginosa, Pseudomonas mallei, Pseudomonas pseudomallei, Rickettsia,Rochalimaia henselae, Salmonella species, preferably S. enteridis, S.typhi and S. derby, and most preferably Salmonella species of the typeSalmonella DT104, Serratia species, preferably S. marcescens, Shigelladysenteriae, S. flexneri, S. boydii and S. sonnei, Spirillum minor,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptobacillus moniliformis, Streptococcus, preferablyS. faecalis, S. faecium and S. durans, Streptococcus agalactiae,Streptococcus pneumoniae, Streptococcus pyogenes (e.g., the Sfb1polypeptide), Treponema carateum, Treponema pallidum, Treponemapertenue, preferably T. pallidum, Ureaplasma urealyticum, Vibriocholerae, Vibrio parahaemolyticus, Yersinia enterocolitica, and Yersiniapestis.

Parasitic haptens or antigens may for example be selected from Malaria(Plasmodium falciparum, P. vivax, P. malariae), Schistosomes,Trypanosomes, Leishmania, Filarial nematodes, Trichomoniasis,Sarcosporidiasis, Taenia (T. saginata, T. solium), Leishmania,Toxoplasma gondii, Trichinelosis (Trichinella spiralis) or Coccidiosis(Eimeria species).

Illustrative fungal haptens or antigens could be derived a fungusselected from Cryptococcus neoformans, Candida albicans, Aspergillusfumigatus and Coccidioidomycosis.

The hapten or antigen may also be derived from any animal, including forexample vertebrates. For example the hapten or antigen may comprisecomponents derived from ovalbumin, keyhole limpet hemocyanin andsperm-whale myoglobulin.

Examples of suitable carriers are those that can increase theimmunogenicity of the conjugate and/or elicit antibodies against thecarrier which are diagnostically, analytically, and/or therapeuticallybeneficial. Useful carriers include polymeric carriers, which can benatural, semi-synthetic or synthetic materials containing one or morefunctional groups, for example primary and/or secondary amino groups,azido groups, hydroxyl groups, or carboxyl groups, to which a reactantmoiety can be attached. The carrier can be water soluble or insoluble,and in some embodiments is a protein or polypeptide. Carriers thatfulfill these criteria are generally known in the art (see, for example,Fattom et al., Infect. Immun. 58:2309-12, 1990; Devi et al., PNAS88:7175-79, 1991; Szu et al., Infect. Immun. 59:4555-61, 1991; Szu etal., J. Exp. Med. 166:1510-24, 1987; and Pavliakova et al., Infect.Immun. 68:2161-66, 2000). A carrier can be useful even if the antibodythat it induces is not of benefit by itself.

Specific, non-limiting examples of water soluble polypeptide carriersinclude, but are not limited to, natural, semi-synthetic or syntheticpolypeptides or proteins from bacteria or viruses. In one embodiment,bacterial products for use as carriers include bacterial wall proteinsand other products (for example, streptococcal or staphylococcal cellwalls), and soluble antigens of bacteria. In another embodiment,bacterial products for use as carriers include bacterial toxins.Bacterial toxins include bacterial products that mediate toxic effects,inflammatory responses, stress, shock, chronic sequelae, or mortality ina susceptible host.

Specific, non-limiting examples of water insoluble carriers include, butare not limited to, aminoalkyl agarose (for example, aminopropyl oraminohexyl SEPHAROSE; Pharmacia Inc., Piscataway, N.J.), aminopropylglass, cross-linked dextran, and the like, to which a reactive moietycan be attached. Other carriers can be used, provided that a functionalgroup is available for covalently attaching a reactive group.

II. Haptens

Disclosed embodiments of such haptens include pyrazoles, particularlynitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureasand thioureas, particularly phenyl ureas, and even more particularlyphenyl thioureas; rotenone and rotenone derivatives, also referred toherein as rotenoids; oxazole and thiazoles, particularly oxazole andthiazole sulfonamides; coumarin and coumarin derivatives; cyclolignans,exemplified by Podophyllotoxin and Podophyllotoxin derivatives; andcombinations thereof.

For the general formulas provided below, if no substituent is indicated,a person of ordinary skill in the art will appreciate that thesubstituent is hydrogen. A bond that is not connected to an atom, but isshown, for example, extending to the interior of a ring system,indicates that the position of such substituent is variable. A curvedline drawn through a bond indicates that some additional structure isbonded to that position, typically a linker or the functional group ormoiety used to couple the hapten to a carrier. Moreover, if nostereochemistry is indicated for compounds having one or more chiralcenters, all enantiomers and diasteromers are included. Similarly, for arecitation of aliphatic or alkyl groups, all structural isomers thereofalso are included.

1. Azoles

A first general class of haptens of the present invention is azoles,typically oxazoles and pyrazoles, more typically nitro oxazoles andnitro pyrazoles, having the following general chemical formula.

With reference to this general formula, R₁-R₄ can be any organic groupthat does not interfere with, and potentially facilitates, the functionas a hapten. More specifically, R₁-R₄ independently are selected from:hydrogen, acyl, aldehydes, alkoxy, aliphatic, particularly loweraliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chainshaving heteroatoms, such as oxygen, nitrogen, sulfur, alkyl,particularly alkyl having 20 or fewer carbon atoms, and even moretypically lower alkyl having 10 or fewer atoms, such as methyl, ethyl,propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide(e.g. —CX₃ where X is a halide, and combinations thereof, either in thechain or bonded thereto), oxime, oxime ether (e.g., methoxyimine,CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly loweralkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such asbenzyl, carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,cyano (—CN), ester, ether, exomethylene, halogen, heteroaryl,heterocyclic, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, andcombinations thereof. Two or more of these R₁-R₄ substituents also maybe atoms, typically carbon atoms, in a ring system bonded or fused tothe compounds having the illustrated general formula. At least one ofthe R₁-R₄ substituents is bonded to a linker or is a functional groupsuitable for coupling to a linker or a carrier molecule. R₁-R₄ mosttypically are aliphatic, hydrogen or nitro groups, even more typicallyalkyl, hydrogen or nitro, and still even more typically lower (10 orfewer carbon atoms) alkyl, hydrogen, nitro, or combinations thereof. Thenumber of nitro groups can vary, but most typically there are 1 or 2nitro groups. X independently is nitrogen or carbon. Y is oxygen, sulfuror nitrogen. If Y is oxygen or sulfur, then there is no R₁ group, andn=0. If Y is nitrogen, then there is at least one R₁ group.

A person of ordinary skill in the art will appreciate that, forcompounds having 2 or more W groups, the relative positions thereof isvariable. For example, a diazole could have nitrogen atoms at the 1 and2 positions, or the 1 and 3 positions. Moreover, more than twoheteroatoms also are possible, such as with triazines.

At least one of R₁-R₄ for these azole compounds is bonded to some othergroup or is a variable functional group. For example, the illustratedcompounds can be coupled either directly to a carrier or to a linker atany of the suitable positions about the azole ring.

Working embodiments typically were mono- or dinitro pyrazolederivatives, such that at least one of R₁-R₄ is a nitro group, andperhaps two of R₁-R₄ are nitro groups, with the remaining R₁-R₄ beingused to couple the hapten to a linker or a carrier.

One particular compound had the following structure.

2. Nitroaryl

A second general class of haptens of the present invention are nitroarylcompounds. Exemplary nitroaryl compounds include, without limitation,nitrophenyl, nitrobiphenyl, nitrotriphenyl, etc., and any and allheteroaryl counterparts, having the following general chemical formula.

With reference to this general formula, such compounds have at leastone, and optionally plural, nitro groups. Thus, at least one of R₁-R₆ isnitro. If more than one of R₁-R₆ is nitro, all combinations of relativering positions of plural nitro substituents, or nitro substituentsrelative to other ring substituents, are included within this class ofdisclosed haptens. Dinitroaryl compounds are most typical. A person ofordinary skill in the art will appreciate that as the number of nitrogroups increases, the number of remaining ring substituents in thegeneral formula decreases. These substituents independently are selectedfrom: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particularly loweraliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chainshaving heteroatoms, such as oxygen, nitrogen, sulfur, alkyl,particularly alkyl having 20 or fewer carbon atoms, and even moretypically lower alkyl having 10 or fewer carbon atoms, such as methyl,ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkylhalide (e.g. —CX₃ where X is a halide, and combinations thereof, eitherin the chain or bonded thereto), oxime, oxime ether (e.g., methoxyimine,CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly loweralkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such asbenzyl, carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, ether, halogen, heteroaryl, hydroxyl,hydroxlyamine, oxime (HO—N═), keto, such as aliphatic ketones, nitro,sulfhydryl, sulfonyl, sulfoxide, exomethylene, and combinations thereof.At least one of the R₁-R₆ substituents is bonded to a linker or is afunctional group suitable for coupling to a linker or a carriermolecule.

Two or more of the R₁-R₆ substituents also may be atoms, typicallycarbon atoms, in a ring system, such as napthalene (shown below) oranthracene type derivatives. Ring systems other than 6-membered ringsystems can be formed, such as fused 6-5 ring systems.

Again, at least one of the ring positions occupied by R₁-R₈ is bonded toa linker or is a variable functional group suitable for coupling, suchas by covalent bonding, to a carrier molecule. For example, nitroarylcompounds of the present invention can include a functional group forcoupling to a carrier, or to a linker, at various optional ringlocations.

Working embodiments are exemplified by nitrophenyl compounds. Solely byway of example, mononitroaryl compounds are exemplified bynitrocinnamide compounds. One embodiment of a nitrocinnamide-basedcompound is exemplified by 4,5-dimethoxy-2-nitrocinnamide, shown below.

The nitrophenyl class of compounds also is represented by dinitrophenylcompounds. At least one of the remaining carbon atoms of the ringpositions not having a nitro group is bonded to a functional group, to alinker, or directly to a carrier. Any and all combinations of relativepositions of these groups are included within the class of disclosedhaptens.

Working embodiments are more particularly exemplified by2,4-dinitrophenyl compounds coupled to a linker, as illustrated below.

R₁-R₃ are as stated above.

3. Benzofurazans

Benzofurazans and derivatives thereof are another class of haptenswithin the scope of the present invention. A general formula for thebenzofurazan-type compounds is provided below.

R₁-R₄ substituents independently are selected from: hydrogen, acyl,aldehydes, alkoxy, aliphatic, particulary lower aliphatic, such asisoprene, substituted aliphatic, heteroaliphatic, e.g., organic chainshaving heteroatoms, such as oxygen, nitrogen, sulfur, alkyl,particularly alkyl having 20 or fewer carbon atoms, and even moretypically lower alkyl having 10 or fewer atoms, such as methyl, ethyl,propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide(e.g. —CX₃where X is a halide, and combinations thereof, either in thechain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine,CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly loweralkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such asbenzyl, carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. Two or more of these R₁-R₄ substituents alsomay be atoms, typically carbon atoms, in a ring system bonded or fusedto the compounds having the illustrated general formula. At least one ofthe R₁-R₄ substituents is bonded to a linker or directly to a carrier. Yis a carbon atom having R₅ and R₆ substituents, where R₅ and R₆ are asstated for R₁-R₄, oxygen or sulfur, typically oxygen.

Compounds where Y is oxygen are more particularly exemplified bycompounds having the following structure, where R₁-R₄ are as statedabove, and most typically are independently hydrogen and lower alkyl.

One working embodiment of a compound according to this class of haptenshad the following chemical structure.

4. Triterpenes

Triterpenes are another class of haptens within the scope of the presentinvention. The basic ring structure common to the cyclic triterpenes hasfour six-membered fused rings, A-D, as indicated below.

A number of publications discuss naturally occurring, semi-synthetic andsynthetic triterpene species within the genus of triterpenes useful forpracticing the present invention, including: J. C. Connolly and R. A.Hill, Triterpenoids, Nat. Prod. Rep., 19, 494-513 (2002); Baglin et al.,A Review of Natural and Modified Beculinic, Ursolic and EchinocysticAcid Derivatives as Potential Antitumor and Anti-HIV Agents, MiniReviews in Medicinal Chemistry, 3, 525-539; W. N. and M. C. Setzer,Plant-Derived Triterpenoids as Potential Antineoplastic Agents, MiniReviews in Medicinal Chemistry, 3, 540-556 (2003); and Baltina, ChemicalModification of Glycyrrhizic Acid as a Route to New Bioactive Compoundsfor Medicine, Current Medicinal Chemistry, 10, 155-171 92003); each ofwhich is incorporated herein by reference. Based on the presentdisclosure and working embodiments thereof, as well as disclosuresprovided by these prior publications, and with reference to this firstgeneral formula, R₁-R₂₁ independently are selected from: hydrogen, acyl,aldehydes, alkoxy, aliphatic, particularly lower aliphatic, such asisoprene, substituted aliphatic, heteroaliphatic, e.g., organic chainshaving heteroatoms, such as oxygen, nitrogen, sulfur, alkyl,particularly alkyl having 20 or fewer carbon atoms, and even moretypically lower alkyl having 10 or fewer atoms, such as methyl, ethyl,propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide(e.g. —CX₃ where X is a halide, and combinations thereof, either in thechain or bonded thereto), oxime, oxime ether (e.g., methoxyimine,CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly loweralkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such asbenzyl, carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. Two or more of these R₁-R₂₁ substituents alsomay be atoms, typically carbon atoms, in a ring system bonded or fusedto the compounds having the illustrated general formula. At least one ofthe R₁-R₂₁ substituents is bonded to a linker or is a functional groupsuitable for coupling to a linker or a carrier molecule. Y is a bond,thereby defining a 5-membered ring, or is a carbon atom bearing R₂₂ andR₂₃ substituents, where these R groups are as stated above.

Disclosed embodiments of triterpenes exemplifying this class of haptensalso may include an E ring, and this E ring can be of various ringsizes, particularly rings having 5-7 atoms, typically carbon atoms, inthe ring. For example, the E ring might be a 6-membered ring, asindicated by the following general formula, where R₁-R₃₁ are as statedabove for R₁-R₂₁.

The following general formula indicates that the R₁₃ substituent may bean acyl group bearing an R₃₃ substituent selected from hydrogen,hydroxyl, ester, i.e. —OR₃₄ where R₃₄ is aliphatic, typically alkyl orsubstituted alkyl, and even more typically lower alkyl, amido, includingprimary amide (—NH₂), secondary amide (—NHR₃₅) and tertiary amide(—NR₃₅R₃₆), where R₃₅ and R₃₆ are aliphatic, typically lower aliphatic,more typically alkyl, substituted alkyl, and even more typically loweralkyl or substituted lower alkyl. This general formula also indicatesthat the R₁ substituent often is an OR₃₂ substituent, where R₃₂ ishydrogen or aliphatic, more typically alkyl or substituted alkyl, andeven more typically lower alkyl. The remaining R groups are as statedabove with reference to the first general formula.

The E ring also may be a 5 membered ring, as indicated by the formulabelow where the R₁-R₂₉ groups are as stated above for R₁-R₂₁.

With reference to these general formulae, the R₁-R₂₉ groups are asstated above for R₁-R₂₁.

As with exemplary compounds where the E ring is a 6-membered ring,compounds where the E ring is a 5-membered ring also can includesubstituents at R₁ and R₁₃ as discussed above. Specifically, thisgeneral formula indicates that the R₁₃ substituent may be an acyl groupbearing an R₃₃ substituent selected from hydrogen, hydroxyl, ester, i.e.—OR₃₄ where R₃₄ is aliphatic, typically alkyl or substituted alkyl, andeven more typically lower alkyl, amido, including primary amide (—NH₂),secondary amide (—NHR₃₅) and tertiary amide (—NR₃₅R₃₆), where R₃₅ andR₃₆ are aliphatic, typically lower aliphatic, more typically alkyl,substituted alkyl, and even more typically lower alkyl or substitutedlower alkyl. This general formula also indicates that the R₁ substituentoften is an OR₃₂ substituent, where R₃₂ is hydrogen or aliphatic, moretypically alkyl or substituted alkyl, and even more typically loweralkyl.

Exemplary compounds also include 5-membered rings as both the A and theE ring. General formulae for such exemplary compounds are providedbelow, where the R₁-R₂₉ substituents are as stated above.

Again, the R₁ and R₁₃ substituents can be oxygen-based functionalgroups. The R₁₃ substituent may be an acyl group bearing an R₃₃substituent selected from hydrogen, hydroxyl, ester, i.e. —OR₃₄ whereR₃₄ is aliphatic, typically alkyl or substituted alkyl, and even moretypically lower alkyl, amido, including primary amide (—NH₂), secondaryamide (—NHR₃₅) and tertiary amide (—NR₃₅R₃₆), where R₃₅ and R₃₆ arealiphatic, typically lower aliphatic, more typically alkyl, substitutedalkyl, and even more typically lower alkyl or substituted lower alkyl.This general formula also indicates that the R₁ substituent often is anOR₃₂ substituent, where R₃₂ is hydrogen or aliphatic, more typicallyalkyl or substituted alkyl, and even more typically lower alkyl.

Exemplary triterpenes of the present invention also may include one ormore sites of unsaturation in one or more of the A-E rings. Exemplarycompounds often have at least one site of unsaturation in the C ring,such as the double bond in the C ring as indicated below.

The site of unsaturation may be an alpha, beta unsaturated ketone, suchas illustrated below for the C ring.

The triterpenes also have a number of stereogenic carbon atoms. A personof ordinary skill in the art will appreciate that particular enantiomersare most likely to occur naturally. While the naturally occurringenantiomer may be most available, and/or effective, for practicingdisclosed embodiments, all other possible stereoisomers are within thescope of the present invention. Moreover, other naturally occurringtriterpenes, or synthetic derivatives thereof, or fully syntheticcompounds, may have (1) different stereochemistry, (2) differentsubstituents, and further may be substituted at positions that are notsubstituted in the naturally occurring compounds. The general formulaeprovided above do not indicate stereochemistry at the chiral centers.This is to signify that both enantiomers at each chiral center, and alldiastereomeric isomer combinations thereof, are within the scope of thepresent invention.

Particular working embodiments of the present invention are exemplifiedby the following general formula, in which the substituents are asstated above.

The stereochemistry and substituents for a naturally occurringtriterpene useful as a hapten for practicing the present invention areshown below.

The hydroxyl group in the A ring typically is oxidized to a carbonylfunctional group in working embodiments. As a result, the carbon atombearing the carbonyl group is no longer a chiral center.

5. Ureas and Thioureas

Ureas and thioureas, particularly aryl and heteroaryl ureas andthioureas, are another class of haptens within the scope of the presentinvention. A general formula for urea-based haptens of the presentinvention is provided below.

With reference to this general formula, R₁-R₃ are independentlyhydrogen, aliphatic, substituted aliphatic, typically alkyl, substitutedalkyl, and even more typically lower alkyl and substituted lower alkyl,cyclic, heterocyclic, aryl and heteroaryl. More specifically, R₁typically is aryl or aliphatic, often having at least one site ofunsaturation to facilitate chromophoric activity. R₂ and R₃ mosttypically are independently hydrogen and lower alkyl. Y is oxygen (ureaderivatives) or sulfur (thioureas).

Aryl derivatives typically have the following formula.

R₁-R₇ independently are selected from: hydrogen, acyl, aldehydes,alkoxy, aliphatic, particularly lower aliphatic, such as isoprene,substituted aliphatic, heteroaliphatic, e.g., organic chains havingheteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkylhaving 20 or fewer carbon atoms, and even more typically lower alkylhaving 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, andbutyl, substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is ahalide, and combinations thereof, either in the chain or bondedthereto), oxime, oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols(i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl)amido, amino, amino acid, aryl, alkyl aryl, such as benzyl,carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. At least one of the R₃-R₇ substituents also isbonded to a linker or to a carrier molecule. Two or more of these R₃-R₇substituents available for such bonding also may be atoms, typicallycarbon atoms, in a ring system bonded or fused to the compounds havingthe illustrated general formula.

Additional rings also can be present, as indicated by the exemplarystructures provided below. The R groups are as stated above for R₁-R₇and Y is oxygen or sulfur.

A particular subclass of thioureas is represented below.

With reference to this general formula, n is 1 to 5, typically 1-2, R₁and R₂ are independently hydrogen or lower alkyl, and X independently isa halide or combinations of different halides.

One example of a working embodiment of a phenyl thiourea is providedbelow.

The trifluoromethyl groups are shown in the 2 and 4 positions relativeto the thiourea moiety. A person of ordinary skill in the art willappreciate that compounds having all relative positions fordisubstituted compounds, such as 2,3, and compounds having more than twotrihaloalkyl substituents, at all possible relative positions of suchplural trihaloalkyl substituents, also are within the scope of thepresent invention. A particular example of a rhodamine thiourea haptenhas the following formula.

6. Rotenones

Rotenone and rotenone-based haptens, collectively referred to asrotenoids, provide another class of haptens within the scope of thepresent invention. A first general formula for rotenone, androtenone-based haptens, is provided below.

A number of publications discuss naturally occurring, semi-synthetic andsynthetic rotenoids that are useful for describing the genus ofrotenoids useful for practicing the present invention, including: LeslieCrombie and Donald Whiting, Biosynthesis in the Rotenoids Group ofNatural Products: Application of Isotope Methodology, Phytochemistry,49, 1479-1507 (1998); and Nianbai Fang, and John Casida, Cube ResinInsecticide: Identification and Biological Activity of 29 RotenoidConstituents; each of which is incorporated herein by reference. Basedon the present disclosure and working embodiments, as well asdisclosures provided by these prior publications, and with reference tothis first general formula, R₁-R₁₄independently are hydrogen, aldehyde,alkoxy, aliphatic, particulary lower aliphatic, such as isoprene,substituted aliphatic, heteroaliphatic, e.g., organic chains havingheteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkylhaving 20 or fewer carbon atoms, and even more typically lower alkylhaving 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, andbutyl, substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is ahalide, and combinations thereof, either in the chain or bonded thereto)amino, amino acid, amido, cyano (—CN), halogen, hydroxyl, hydroxylamine,oxime (HO—N═), oxime ether (e.g., methoxyimine, CH₃—O—N═) alkylhydroxyl, particularly lower alkyl hydroxyl, carbonyl, keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, carboxyl,carboxylate (and salts thereof, such as Group I metal or ammonium ioncarboxylates) ester, alkyl ester, acyl, exomethylene, ether, cyclic,heterocyclic, aryl, alkyl aryl, such as benzyl, heteroaryl,polysaccharides, carbohydrate, monosaccharides, such as glucose andfructose, disaccharides, such as sucrose and lactose, oligosaccharidesand polysaccharides, and combinations thereof. Two or more of theseR₁-R₁₄ substituents also may be atoms, typically carbon atoms, in a ringsystem bonded or fused to the compounds having the illustrated generalformula. At least one of the R₁-R₁₄ substituents also is bonded to alinker or to a carrier molecule.

While R₆ and R₇ can be as stated above, such substituents more typicallyindependently are hydrogen, OR₁₅, where R₁₅ is hydrogen, aliphatic,substituted aliphatic, typically alkyl, substituted alkyl, and even moretypically lower alkyl and substituted lower alkyl, such as lower alkylhalides, cyclic, heterocyclic, aryl and heteroaryl, —NR₂₁, where R₂₁ ishydrogen, aliphatic, substituted aliphatic, typically alkyl, substitutedalkyl, and even more typically lower alkyl and substituted lower alkyl,such as lower alkyl halides, cyclic, heterocyclic, aryl and heteroaryl,or N-L-RG, where L is a linker or a reactive group, such as an amine, asdiscussed in more detail herein.

R₆ and R₇ also can form a double bond, such as a double bond to anoxygen to form a carbonyl. If R₆ and/or R₇ are not -L-RG, then at leastone of the R substituents is bonded to a linker or to a carriermolecule.

The B ring also can include at least one additional site ofunsaturation. For example, R₅ and R₁₂ can form a double bond.

R₁₀ and R₁₁ can be joined in a 5- or 6-membered ring. For example, R₁₀and R₁₁ may define a pyran or furan ring, and more particularly is asubstituted and/or unsaturated pyran or furan ring.

Certain exemplary rotenone-based haptens of the present invention alsotypically satisfy the following second general formula.

With reference to this second general formula, the R substituents are asstated above. If R₆ or R₇ is not -L-RG, then at least one of theremaining R groups is bonded to a linker or to a carrier.

R₁₀ and R₁₁ can be joined in a 5- or 6-membered ring, such as a pyran orfuran, and more particularly a substituted and/or unsaturated pyran orfuran ring. Thus, a third general formula useful for describing certainrotenone-based haptens of the present invention is provided below, wherethe R substituents are as stated above.

Y is a bond, thereby defining a 5-membered ring, or is a carbon atom ina 6-membered ring bearing R₁₉ and R₂₀ substituents, as shown below,where the R substituents are as stated above.

R₅ and R₁₂ at the ring juncture are shown without indicating particularstereochemistry. The naturally occurring compound has a cis-ringjuncture, but racemic mixtures also are useful for practicing thepresent invention. Also, the trans stereoisomer likely quicklyequilibrates to form the racemic mixture.

Working embodiments of compounds within this class more typicallysatisfy the following third general formula.

With reference to this general formula, R₆ and R₇ are hydrogen, alkyl,or define a double bond, such as to oxygen to form a carbonyl. R₁₅ andR₁₆ independently are hydrogen and aliphatic, typically lower aliphatic,such as alkenyl, one example of which is isoprene, as shown below.

Again, a particular enantiomer is shown in the above formula, but aperson of ordinary skill in the art will appreciate that the scope ofthe present invention is not limited to the particular enantiomer shown.Instead, all stereoisomers that act as haptens also are within the scopeof the disclosure. All substitutions discussed above for this class ofcompounds applies to this particular compound. Other substitutions alsoare readily apparent to a person of ordinary skill in the art. Forexample, the methoxy groups on the A ring can be any alkoxy compound,particular lower alkoxy groups. The isoprene unit also provides anolefin that can be synthetically modified, perhaps to provide analternative position, or at least a second position, for coupling thehapten to a linker or a carrier molecule. For example, the olefin couldbe converted to an alcohol by hydroboration. It also could be convertedto a halide or an epoxide either for use as a hapten or as intermediatesuseful for further transformation.

A fourth general formula for describing rotenone-based haptens of thepresent invention is particularly directed to rotenone isoxazolines, asprovided below.

R-R₅ independently are hydrogen, aldehyde, alkoxy, aliphatic,particularly lower aliphatic, including all branched chain isomers, suchas isoprene, and all stereoisomers, substituted aliphatic,heteroaliphatic, e.g., organic chains having heteroatoms, such asoxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewercarbon atoms, and even more typically lower alkyl having 10 or feweratoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substitutedalkyl, such as alkyl halide (e.g. —CX₃ where X is a halide, andcombinations thereof, either in the chain or bonded thereto) amino,amino acid, amido, cyano (—CN), halogen, hydroxyl, hydroxylamine, oxime(HO—N═), oxime ether (e.g., methoxyimine, CH₃—O—N═) alkyl hydroxyl,particularly lower alkyl hydroxyl, carbonyl, keto, such as aliphaticketones, nitro, sulfhydryl, sulfonyl, sulfoxide, carboxyl, carboxylate(and salts thereof, such as Group I metal or ammonium ion carboxylates)ester, alkyl ester, acyl, exomethylene, ether, cyclic, heterocyclic,aryl, alkyl aryl, such as benzyl, heteroaryl, polysaccharides,carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, and combinations thereof. At least one of the R-R₅substituents also is bonded to a linker or to a carrier molecule. Y isoxygen, nitrogen, or sulfur.

A particular working embodiment of a rotenone-based hapten satisfyingthis fourth general formula is provided below.

7. Oxazoles and Thiazoles

Oxazole and thiazole sulfonamides provide another class of haptenswithin the scope of the present invention. A general formula for oxazoleand thiazole sulfonamides is provided below.

With reference to this first general formula R₁-R₃ independently areselected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic,particularly lower aliphatic, such as isoprene, substituted aliphatic,heteroaliphatic, e.g., organic chains having heteroatoms, such asoxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewercarbon atoms, and even more typically lower alkyl having 10 or feweratoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substitutedalkyl, such as alkyl halide (e.g. —CX₃ where X is a halide, andcombinations thereof, either in the chain or bonded thereto), oxime,oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic oralkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, aminoacid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides,such as glucose and fructose, disaccharides, such as sucrose andlactose, oligosaccharides and polysaccharides, carbonyl, carboxyl,carboxylate (including salts thereof, such as Group I metal or ammoniumion carboxylates), cyclic, heterocyclic, cyano (—CN), ester, alkylester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime(HO—N═), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl,sulfoxide, exomethylene, and combinations thereof. Two or more of theseR₁-R₃ substituents also may be atoms, typically carbon atoms, in a ringsystem bonded or fused to the compounds having the illustrated generalformula. At least one of the R₁-R₃ substituents is bonded to a linker oris a functional group suitable for coupling to a linker or a carriermolecule. Y is oxygen or sulfur, typically sulfur.

For certain exemplary working embodiments, R₁ has been amido, such asthe amide derivatives shown below. R₂ provides a position for couplingto a linker or to a carrier molecule, although the positions indicatedby R₁ and R₂ also provide alternative or additional positions forcoupling to a linker and/or carrier molecule. R₂, for certain workingembodiments, has been —SO₂, and has been used to couple linkers byforming a sulfonamide. Thus, a second general formula for workingembodiments of haptens exemplifying this class of haptens is indicatedbelow, where the R₃-R₆ substituents and Y are as stated above.

For certain working embodiments R₆ has been alkyl, particularly loweralkyl, such as methyl, and Y has been sulfur.

One working embodiment of a compound according to this class of haptenshad the following chemical structure.

The thiazole or oxazole might also be part of a larger ring system. Forexample, the 5-membered oxazole or thiazole might be coupled to at leastone additional ring, such as a phenyl ring, as indicated below.

While the R₁-R₅ groups generally can be as stated above, such compoundsalso provide a position for coupling to a linker and/or to a carriermolecule, such as a R₅. One possible sulfonamide derivative is providedbelow.

8. Coumarins

Coumarin and coumarin derivatives provide another class of haptenswithin the scope of the present invention. A general formula forcoumarin and coumarin derivatives is provided below.

With reference to this general formula, R₁-R₆ independently are selectedfrom: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particulary loweraliphatic, such as isoprene, substituted aliphatic, heteroaliphatic,e.g., organic chains having heteroatoms, such as oxygen, nitrogen,sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, andeven more typically lower alkyl having 10 or fewer atoms, such asmethyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such asalkyl halide (e.g. —CX₃ where X is a halide, and combinations thereof,either in the chain or bonded thereto,), oxime, oxime ether (e.g.,methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl,particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkylaryl, such as benzyl, carbohydrate, monosaccharides, such as glucose andfructose, disaccharides, such as sucrose and lactose, oligosaccharidesand polysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. At least one of the R₁-R₆substituents alsotypically is bonded to a linker or a carrier molecule. Certain workingembodiments have used the position indicated as having an R₅ substituentfor coupling to a linker or carrier molecule. The 4 position can beimportant if fluorescence is used to detect these compounds.Substituents other than hydrogen at the 4 position are believed toquench fluorescence, although such derivatives still may bechromophores. Y is oxygen, nitrogen or sulfur. Two or more of the R₁-R₆substituents available for forming such compounds also may be atoms,typically carbon atoms, in a ring system bonded or fused to thecompounds having the illustrated general formula. Exemplary embodimentsof these types of compounds are provided below.

A person of ordinary skill in the art will appreciate that the ringsalso could be heterocyclic and/or heteroaryl.

Working embodiments typically were fused A-D ring systems having atleast one carrier molecule coupling position, with one possible couplingposition being indicated below.

With reference to this general formula, the R and Y variable groups areas stated above. Most typically, R₁-R₁₄ independently are hydrogen orlower alkyl. Particular embodiments of coumarin-based haptens include2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carboxylicacid

and diethyl coumarin

9. Cyclolignans

Lignin-based compounds, particulary cyclolignans, such asPodophyllotoxin and derivatives thereof, provide another class ofhaptens within the scope of the present invention. A first generalformula for these cyclolignin-based derivatives is provided below.

A number of publications discuss naturally occuring, semi-synthetic andsynthetic cyclolignans that are useful for describing the genus ofcyclolignans useful for practicing the present invention, including:Stephanie Desbene and Sylviane Giorgi-Renault, Drugs that InhibitTubulin Polymerization: The Particular Case of Podophyllotoxin andAnalogues, Curr. Med. Chem. —Anti-Cancer Agents, 2, 71-90 (2002); M.Gordaliza et al., Podophyllotoxin: Distribution, Sources, Applicationsand New Cytotoxic Derivatives, Toxicon, 44, 441-459 (2004); PhillipeMeresse et al., Etoposide: Discovery and Medicinal Chemistry, CurrentMedicinal Chemistry, 11, 2443-2466 (2004); M. Pujol et al., Synthesisand Biological Activity of New Class of Dioxygenated Anticancer Agents,Curr. Med. Chem. —Anti-Cancer Agents, 5, 215-237 (2005); and YoungjaeYou, Podophyllotoxin Derivatives: Current Synthetic Approaches for NewAnticancer Agents, Current Pharmaceutical Design, 11, 1695-1717(2005);each of which is incorporated herein by reference. Based on the presentdisclosure and working embodiments, as well as disclosures provided bythese prior publications, and with reference to this first generalformula, R₁-R₁₂ typically are selected from hydrogen, aldehyde, alkoxy,aliphatic, particulary lower aliphatic, such as isoprene, substitutedaliphatic, heteroaliphatic, e.g., organic chains having heteroatoms,such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 orfewer carbon atoms, and even more typically lower alkyl having 10 orfewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl,substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is a halide,and combinations thereof, either in the chain or bonded thereto,) amino,amino acid, amido, cyano (—CN), halogen, hydroxyl, hydroxylamine, oxime,oxime ether (e.g., methoxyimine, CH₃—O—N═) alkyl hydroxyl, particularlylower alkyl hydroxyl, carbonyl, keto, such as aliphatic ketones, nitro,sulfhydryl, sulfonyl, sulfoxide, carboxyl, carboxylate (and saltsthereof, such as Group I metal or ammonium ion carboxylates) ester,alkyl ester, acyl, exomethylene, ether, cyclic, heterocyclic, aryl,alkyl aryl, such as benzyl, heteroaryl, polysaccharides, carbohydrate,monosaccharides, such as glucose and fructose, disaccharides, such assucrose and lactose, oligosaccharides and polysaccharides, andcombinations thereof. At least one of R₁-R₁₂ provides a position forcoupling the compound to a linker or to a carrier molecule. Furthermore,certain of the R groups may be atoms in a ring system. For example, R₂and R₃, as well as two of R₇-R₁₀, can be joined together in a ringsystem. At least one of R₁₂ and R₁₁ also often is an aryl group, such asa benzene ring or a substituted benzene ring.

Certain working embodiments also satisfied the following second generalformula, where the R substituents are as stated above.

Exemplary compounds where at least one of R₁₁ and R₁₂ is an aryl grouphave the following general formula, where the R substituents are asstated above.

R₁₆-R₂₀ are generally as stated above, but more typically independentlyare hydrogen or alkoxy, typically lower alkoxy, such as methoxy, asshown below.

At least one of the R substituents typically is bonded to a linker, is areactive functional group capable of reacting with a linker, or is-L-RG. For example, R₅ often is -L-RG.

R₅ and R₆ also may form a double bond, such as a double bond to oxygento form a carbonyl functional group or a double bond to a nitrogen atomto form an imine. Certain exemplary compounds where R₅ and R₆ form adouble bond had the following general formula, where the remaining Rsubstituents are as stated above. Y is selected from nitrogen, oxygen orsulfur. If Y is nitrogen, then the nitrogen atom may further have bondedthereto hydrogen, or some atom, functional group or chemical moietyother than hydrogen. For example, the nitrogen may have an aliphaticsubstituent, such an alkyl group, an aryl or heteroaryl substituent, ora substituted aryl or heteroaryl substituent, such as alkyl and/oralkoxy substituted aryl or heteroaryl substituent.

R₁₆-R₂₀ independently are selected from hydrogen and alkoxy, moretypically lower alkoxy, such as methoxy, as indicated below.

As with all hapten conjugates of the present invention, at least one ofthe R substituents typically is bonded to a linker, is a reactivefunctional group capable of reacting with a linker, is -L-RG, or isdirectly bonded to a carrier. For example, R₉ often is -L-RG.

The chemical structure for Podophyllotoxin, a compound exemplifying thiscyclolignan class of haptens, is provided below.

Podophyllotoxin, also referred to as podofilox, is a non-alkaloid toxinhaving a molecular weight of 414.40 and a compositional formula ofC₂₂H₂₂O₈. Podophyllotoxin is present at concentrations of 0.3 to 1.0% bymass in the rhizome of American Mayapple Podophyllum peltatum. Themelting point of Podophyllotoxin is 183.3-184.0° C.

Accordingly, cyclolignans according to the present invention basedsubstantially on the Podophyllotoxin structure have the followinggeneral formula, where Y is selected from nitrogen, oxygen or sulfur.

A specific example of a cyclolignan hapten according to the presentinvention is shown below.

This compound was made starting with Podophyllotoxin. The hydroxyl groupof Podophyllotoxin was oxidized to a ketone. The ketone was then reactedwith a substituted hydrazine to produce the compound indicated above.The hydrazine reagent can be substituted as desired, including aliphaticand aryl substituents.

10. Heterobiaryl

Another general class of haptens of the present invention isheterobiaryl compounds, typically phenyl quinolines and quinoxalines.Disclosed heterobiaryl compounds have a first general chemical formulaas below.

With reference to this general formula, A-D are selected from carbon,nitrogen, oxygen, and sulfur, and any and all combinations thereof. Mosttypically A-D are carbon or nitrogen. R₁-R₂ substituents independentlyare selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic,particularly lower aliphatic, substituted aliphatic, heteroaliphatic,e.g., organic chains having heteroatoms, such as oxygen, nitrogen,sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, andeven more typically lower alkyl having 10 or fewer atoms, such asmethyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such asalkyl halide (e.g. —CX₃ where X is a halide, and combinations thereof,either in the chain or bonded thereto), oxime, oxime ether (e.g.,methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl,particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkylaryl, such as benzyl, alkoxy aryl, such as methoxy and ethoxy,carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. Two or more of the R₁-R₂ substituents, mosttypically plural R₁ substituents, also may be atoms, typically carbonatoms, in a ring system bonded or fused to the compounds having theillustrated general formula. At least one of the R₁-R₂ substituentstypically is bonded to a linker or directly to a carrier.

Particular embodiments of the heterobiaryl compounds have the followingformula.

R1 and R2 are as stated above for the first general formula. Y isoxygen, nitrogen or sulfur, typically nitrogen. If Y is nitrogen, thenthe formula also can include double bonds to the one or more nitrogenatoms.

Compounds having a single heteroatom are exemplified byphenylquinolines, such as follows.

More particular embodiments include aryl substituted haptens,exemplified by the following general formula.

With reference to this general formula, R₁-R₃ are as indicated above.More typically, R₁ is hydrogen, R₂ is acyl, and R₃ is alkoxy. Aparticular example, 2-(3,4-dimethoxyphenyl)quinoline-4-carboxylic acid,is provided below.

Compounds having two heteroatoms are represented by quinoxalines, asindicated by the general formula below.

A particular example of biaryl-diheteroatom hapten of the presentinvention is exemplified by 3-hydroxy-2-quinoxalinecarbamide, below.Again, the R₁ and R₂ substituents are as stated above with respect tothis class of haptens.

11. Azoaryl

Another general class of haptens of the present invention is azoarylcompounds, such as azobenzenes, having a first general chemical formulaas below.

R₁-R₂ substituents independently are selected from: hydrogen, acyl,aldehydes, alkoxy, aliphatic, particularly lower aliphatic, substitutedaliphatic, heteroaliphatic, e.g., organic chains having heteroatoms,such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 orfewer carbon atoms, and even more typically lower alkyl having 10 orfewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl,substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is a halide,and combinations thereof, either in the chain or bonded thereto,),oxime, oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e.aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido,amino, amino acid, aryl, alkyl aryl, such as benzyl, alkoxy aryl, suchas methoxy and ethoxy, carbohydrate, monosaccharides, such as glucoseand fructose disaccharides, such as sucrose and lactose,oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate(including salts thereof, such as Group I metal or ammonium ioncarboxylates), cyclic, heterocyclic, cyano (—CN), ester, alkyl ester,ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═),keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide,sulfonyl, exomethylene, and combinations thereof. Two or more R₂substituents also may be atoms, typically carbon atoms, in a ring systembonded or fused to the compounds having the illustrated general formula.For example, 2 R₂ substituents may form a fused phenyl ring, or a fusedheterocyclic or heteroaryl structure.

Certain disclosed azoaryl compounds have a first amine substituent and asecond aryl substituent. These compounds typically have the followingformula.

With reference to this general formula, R₂-R₄ are as stated above withrespect to this class of haptens, with particular embodiments havingR₂-R₃ aliphatic, particularly alkyl, more particularly lower alkyl, andR₄ hydrogen.

A third general formula for describing azoaryl compounds is providedbelow.

R₂-R₅ are as stated above for this particular class of haptens. At leastone of R₂-R₅ defines a position for coupling a linker or carrier to theazoaryl hapten to form a conjugate. For example, R₅ may be a sulfonylhalide functional group. Sulfonyl halides, such as that shown below, areuseful functional groups for coupling linkers to the azoaryl haptens.

With reference to this formula, R₂-R₅ are as stated above. X is ahalide. A particular embodiment of these azoaryl haptens,4-(dimethylamino)azobenzene-4′-sulfonyl chloride, has the formulaprovided below.

12. Benzodiazepines

Another class of haptens according to the present invention is thebenzodiazepine haptens, having a first general formula as indicatedbelow.

R₁-R₅ independently are selected from: acyl, aldehydes, alkoxy,aliphatic, particularly lower aliphatic, substituted aliphatic,heteroaliphatic, e.g., organic chains having heteroatoms, such asoxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewercarbon atoms, and even more typically lower alkyl having 10 or feweratoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substitutedalkyl, such as alkyl halide (e.g. —CX₃ where X is a halide, andcombinations thereof, either in the chain or bonded thereto), oxime,oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic oralkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, aminoacid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides,such as glucose and fructose, disaccharides, such as sucrose andlactose, oligosaccharides and polysaccharides, carbonyl, carboxyl,carboxylate (including salts thereof, such as Group I metal or ammoniumion carboxylates), cyclic, cyano (—CN), ester, ether, exomethylene,halogen, heteroaryl, heterocyclic, hydrogen, hydroxyl, hydroxylamine,oxime (HO—N═), keto, such as aliphatic ketones, nitro, sulfhydryl,sulfonyl, sulfoxide, and combinations thereof. Two or more of the R₅substituents also may be atoms, typically carbon atoms, in a ring systembonded or fused to the compounds having the illustrated general formula.At least one of the R₁-R₅ positions is bonded to a linker or is occupiedby a functional group suitable for coupling to a linker or a carriermolecule. R₁-R₅ most typically are aliphatic, aryl, hydrogen, orhydroxyl, even more typically alkyl, hydrogen or phenyl. Y is oxygen orsulfur, most typically oxygen.

Particular embodiments of the benzodiazepine haptens have R₁ aryl, asindicated below.

For these embodiments, R₂-R₅ are as stated above for this class ofhaptens, more typically such substituents are independently selectedfrom aliphatic, particular alkyl, hydrogen and hydroxyl. Certaindisclosed embodiments are phenyl compounds, as illustrated below.

Again, R₂-R₆ are as stated above, but more typically such substituentsare independently selected from aliphatic, particularly alkyl, hydrogenand hydroxyl. Certain disclosed embodiments are phenyl compounds, asillustrated below. A particular embodiment,4-(2-hydroxyphenyl)-1H-benzo[b][1,4]diazepine-2(3H)-one, is providedbelow.

III. Linkers

1. General

As indicated by the general formulahapten-optional linker-carrierconjugates of the present application may include linkers. Any linkercurrently known for this purpose, or developed in the future, can beused to form conjugates of the present invention by coupling to thehaptens disclosed herein. Useful linkers can either be homo- orheterobifunctional, but more typically are heterobifunctional.

2. Aliphatic

Solely by way of example, and without limitation, a first class oflinkers suitable for forming disclosed hapten conjugates are aliphaticcompounds, such as aliphatic hydrocarbon chains having one or more sitesof unsaturation, or alkyl chains. The aliphatic chain also typicallyincludes terminal functional groups, including by way of example andwithout limitation, a carbonyl-reactive group, an amine-reactive group,a thiol-reactive group or a photo-reactive group, that facilitatecoupling to haptens and other desired compounds, such as specificbinding moieties. The length of the chain can vary, but typically has anupper practical limit of about 30 atoms. Chain links greater than about30 carbon atoms have proved to be less effective than compounds havingsmaller chain links. Thus, aliphatic chain linkers typically have achain length of from about 1 carbon atom to about 30 carbon atoms.However, a person of ordinary skill in the art will appreciate that, ifa particular linker has greater than 30 atoms, and still operatesefficiently for linking the hapten to a carrier molecule coupling unit,and the conjugate still functions as desired, then such chain links arestill within the scope of the present invention.

3. Alkylene Oxides

A second class of linkers useful for practicing the present inventionare the alkylene oxides. The alkylene oxides are represented herein byreference to glycols, such as ethylene glycols. Hapten conjugates of thepresent invention have proved particularly useful if the hydrophilicityof the linker is increased relative to their hydrocarbon chains. As aresult, the alkylene oxides, such as the glycols, have proved useful forpracticing this invention. A person of ordinary skill in the art willappreciate that, as the number of oxygen atoms increases, thehydrophilicity of the compound also may increase. Thus, linkers of thepresent invention typically have a formula of (—OCH₂CH₂O—)_(n) where nis from about 2 to about 15, but more particularly is from about 2 toabout 8.

Heterobifunctional polyalkyleneglycol linkers useful for practicingcertain disclosed embodiments of the present invention are described inassignee's co-pending applications, including “Nanoparticle Conjugates,”U.S. patent application Ser. No.11/413,778, filed Apr. 28, 2006;“Antibody Conjugates,” U.S. application Ser. No. 11/413,415, filed Apr.27, 2006; and “Molecular Conjugate,” U.S. Provisional Patent ApplicationNo. 60/739,794, filed Nov. 23, 2005; all of which applications areincorporated herein by reference. A person of ordinary skill in the artwill appreciate that the linkers disclosed in these applications can beused to link specific binding moieties, signal generating moieties andhaptens in any and all desired combinations. Heterobifunctionalpolyalkyleneglycol linkers are disclosed below, and their useexemplified by reference to coupling specific binding moieties, such asantibodies and nucleic acids, to haptens and detectable labels. Inparticular, conjugates of anti-hapten antibodies and detectable labelsand conjugates of primary antibodies or nucleic acids with haptens areexemplified below.

One particular embodiment of a linker for use with disclosed conjugatesis a heterobifunctional polyalkyleneglycol linker having the generalstructure shown below:A

(CH₂)_(x)—O

_(y)Bwherein A and B include different reactive groups, x is an integer from2 to 10 (such as 2, 3 or 4), and y is an integer from 1 to 50, forexample, from 2 to 30 such as from 3 to 20 or from 4 to 12. One or morehydrogen atoms can be substituted for additional functional groups suchas hydroxyl groups, alkoxy groups (such as methoxy and ethoxy), halogenatoms (F, Cl, Br, I), sulfato groups and amino groups (including mono-and di-substituted amino groups such as dialkyl amino groups.

A and B of the linker can independently include a carbonyl-reactivegroup, an amine-reactive group, a thiol-reactive group or aphoto-reactive group, but are not the same. Examples ofcarbonyl-reactive groups include aldehyde- and ketone-reactive groupslike hydrazine derivatives and amines. Examples of amine-reactive groupsinclude active esters such as NHS or sulfo-NHS, isothiocyanates,isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals,epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydridesand the like. Examples of thiol-reactive groups includenon-polymerizable Michael acceptors, haloacetyl groups (such asiodoacetyl), alkyl halides, maleimides, aziridines, acryloyl groups,vinyl sulfones, benzoquinones, aromatic groups that can undergonucleophilic substitution such as fluorobenzene groups (such as tetraand pentafluorobenzene groups), and disulfide groups such as pyridyldisulfide groups and thiols activated with Ellman's reagent. Examples ofphoto-reactive groups include aryl azide and halogenated aryl azides.Alternatively, A and/or B can be a functional group that reacts with aspecific type of reactive group. For example, A and/or B can be an aminegroup, a thiol group, or a carbonyl-containing group that will reactwith a corresponding reactive group (such as an amine-reactive group,thiol-reactive group or carbonyl-reactive group, respectively) that hasbeen introduced or is otherwise present on a hapten and/or a carrier.Additional examples of each of these types of groups will be apparent tothose skilled in the art. Further examples and information regardingreaction conditions and methods for exchanging one type of reactivegroup for another are provided in Hermanson, “Bioconjugate Techniques,”Academic Press, San Diego, 1996, which is incorporated by referenceherein. In a particular embodiment, a thiol-reactive group is other thanvinyl sulfone.

In some embodiments, a thiol-reactive group of the heterobifunctionallinker is covalently attached to a specific-binding moiety and anamine-reactive group of the heterobifunctional linker is covalentlyattached to an amine-reactive group of a hapten derivative (such as anactivated ester formed by reacting a carboxylic acid group with SMCC),the nanoparticle, or vice versa. For example, a thiol-reactive group ofthe heterobifunctional linker can be covalently attached to a cysteineresidue (such as following reduction of cystine bridges) of thespecific-binding moiety or a thiol-reactive group of theheterobifunctional linker can be covalently attached to a thiol groupthat is introduced to the specific-binding moiety, and theamine-reactive group is attached to an activated hapten derivativehaving an amine reactive group such as an activated ester. Where theconjugate includes an anti-hapten antibody conjugated to a detectablelabel, a thiol-reactive group of the heterobifunctional linker can becovalently attached to the antibody and an amine reactive group of theheterobifunctional linker can be covalently attached to the antibody andan amine reactive group of the heterobifunctional linker can becovalently attached to the detectable label or vice versa.

Alternatively, an aldehyde-reactive group of the heterobifunctionallinker can be covalently attached to a specific binding moiety and aeither a functional group or a different reactive group of the linker isattached to a hapten. Where the specific binding moiety is ananti-hapten antibody and the antibody is conjugated to a detectablelabel, an aldehyde-reactive group of the heterobifunctional linker canbe covalently attached to the antibody and an amine-reactive group ofthe heterobifunctional linker can be covalently attached to thedetectable label, or vice versa. In a particular embodiment, analdehyde-reactive group of the heterobifunctional linker can becovalently attached to an aldehyde formed on a glycosylated portion ofan anti-hapten antibody, and an amine-reactive group of the linker isattached to the detectable label. In yet other embodiments, analdehyde-reactive group of the heterobifunctional linker is covalentlyattached to the anti-hapten antibody and a thiol-reactive group of theheterobifunctional linker is attached to the detectable label, or viceversa. In yet other embodiments, an aldehyde-reactive group of theheterobifunctional linker is covalently attached to the specific-bindingmoiety and a thiol-reactive group of the heterobifunctional linker isattached to the nanoparticle, or vice versa.

In some embodiments the heterobifunctional linker has the formula:A-X

(CH₂)_(x)—O

_(y)Y—Bwherein A and B are different reactive groups and are as stated above; xand y are as stated above, and X and Y are additional spacer groups, forexample, spacer groups having between 1 and 10 carbons such as between 1and 6 carbons or between 1 and 4 carbons, and optionally containing oneor more amide linkages, ether linkages, ester linkages and the like.Spacers X and Y can be the same or different, and can bestraight-chained, branched or cyclic (for example, aliphatic or aromaticcyclic structures), and can be unsubstituted or substituted. Functionalgroups that can be substituents on a spacer include carbonyl groups,hydroxyl groups, halogen (F, Cl, Br and I) atoms, alkoxy groups (such asmethoxy and ethoxy), nitro groups, and sulfate groups.

In particular embodiments, the heterobifunctional linker comprises aheterobifunctional polyethylene glycol linker having the formula:

wherein n=1 to 50, for example, n=2 to 30 such as n=3 to 20 or n=4 to12. In more particular embodiments, a carbonyl of a succinimide group ofthis linker is covalently attached to an amine group on a detectablelabel and a maleimide group of the linker is covalently attached to athiol group of an anti-hapten antibody, or vice versa. In other moreparticular embodiments, an average of between about 1 and about 10specific-binding moieties are covalently attached to a nanoparticle,such as semiconductor nanocrystals (such as quantum dots, obtained forexample, from Invitrogen Corp., Eugene, Oreg.; see, for example, U.S.Pat. Nos. 6,815,064, 6,682,596 and 6,649,138, each of which patents isincorporated by reference herein), paramagnetic nanoparticles, metalnanoparticles, and superparamagnetic nanoparticles.

In other particular embodiments, the heterobifunctional linker comprisesa heterobifunctional polyethylene glycol linker having the formula:

wherein m=1 to 50, for example, m=2 to 30 such as m=3 to 20 or m=4 to12. In more particular embodiments, a hydrazide group of the linker iscovalently linked with an aldehyde group of an antihapten antibody and amaleimide group of the linker is covalently linked with a thiol group ofa detectable label, or vice versa. In even more particular embodiments,the aldehyde group of the specific-binding moiety is an aldehyde groupformed in an Fc portion of an anti-hapten antibody by oxidation of aglycosylated region of the Fc portion of the antibody. In other evenmore particular embodiments, an average of between about 1 and about 10anti-hapten antibodies are covalently attached to a nanoparticle.Briefly, maleimide/hydrazide PEG-linkers of the formula above can besynthesized from corresponding maleimide/active ester PEG linkers (whichare commercially available, for example, from Quanta Biodesign, Powell,Ohio) by treatment with a protected hydrazine derivative (such as aBoc-protected hydrazine) followed by treatment with acid.

A conjugate of a specific binding moiety (SBM) and one or more of thedisclosed haptens is provided. The SBM in these conjugates can include,for example, an antibody, a nucleic acid, a lectin or an avidin such asstreptavidin. If the SBM includes an antibody, the antibody canspecifically bind any particular molecule or particular group of highlysimilar molecules, for example, an antibody that specifically binds aparticular protein that may be present in a sample. Alternatively, theantibody can be an anti-antibody antibody that can be used as asecondary antibody in an immunoassay. For example, the antibody cancomprise an anti-IgG antibody such as an anti-mouse IgG antibody, ananti-rabbit IgG antibody or an anti-goat IgG antibody.

In particular embodiments, a disclosed antibody conjugate has theformula:

wherein Ab is an antibody, n=1 to 50 (such as n=2 to 30, n=3 to 20 orn=4 to 12) and j=1 to 10 (such as j=2 to 6 or j=3 to 4). X is a spacergroup suitable for spacing the hapten from the remainder of theconjugate and allowing the hapten to be coupled to the remainder of theconjugate. For example, a spacer group may be an aliphatic or aromaticgroup, typically an aliphatic group, and even more typically an alkyl orsubstituted alkyl group having from about 1 to about 10 carbon atoms,such as between 1 and 6 carbons or between 1 and 4 carbons. The spaceralso may include atoms other than carbon, such as heteroatoms, includingbut not limited to, halides, nitrogen, oxygen, sulfur, and combinationsthereof. Such additional atoms can define functional groups. Forexample, the spacer group optionally may include one or more amidelinkages, ether linkages, ester linkages, amine linkages and the like.The structure of the spacer will depend on the chemistry used to couplethe hapten to the linker, and specific examples of such linkages arelater discussed with regard to specifically disclosed haptens, but ingeneral the group X can, for example, be formed by reacting an amine onthe linker with an amine reactive group added to the hapten (or viceversa) or a carbonyl on the linker with a carbonyl reactive group addedto the hapten (or vice versa).

Alternatively a conjugate of an antibody with one or more of thedisclosed haptens can have the following formula:

wherein Ab is an antibody, m=1 to 50 (such as m=2 to 30, m=3 to 20 orn=4 to 12) and k=1 to 10 (such as k=2 to 6 or kj=3 to 4) and X is againa spacer group, for example, a spacer group having between 1 and 10carbon atoms, such as between 1 and 6 carbons or between 1 and 4carbons, and optionally containing one or more amide linkages, etherlinkages, ester linkages, amine linkages and the like.

In other embodiments, the specific binding moiety linked to one or morehaptens is a nucleic acid. In a particular embodiment, such a conjugatecan have the formula:

wherein Nuc is any nucleic acid base containing compound, including anucleoside, nucleotide, nucleotide phosphate (such as a nucleotidetriphosphate), an oligonucleotide, or a polynucleotide, and m can be,for example, from about 1 to 500 such as m=1 to 100 or m=1 to 50) and Xis yet again a spacer group, for example, a spacer group having between1 and 10 carbon atoms, such as between 1 and 6 carbons or between 1 and4 carbons, and optionally containing one or more amide linkages, etherlinkages, ester linkages, amine linkages and the like.

Also provided is a conjugate of an antibody that specifically binds adisclosed hapten. In particular embodiments, such a conjugate can havethe following formula:

wherein AHAb is an anti-hapten antibody, DL is a detectable label suchas an enzyme, n=1 to 50 (such as n=2 to 30, n=3 to 20 or n=4 to 12) ando=1 to 10 (such as o=2 to 6 or o=3to 4); or

wherein AHAb is an anti-hapten antibody, DL is a detectable label suchas a nanoparticle, n=1 to 50 (such as n=2 to 30, n=3 to 20 or n=4 to 12)and p=1 to 10 (such as p=2 to 6 or p=3 to 4).

In yet other particular embodiments, a disclosed conjugate comprises aconjugate having the formula:

wherein AHAb is an anti-hapten antibody, DL is a detectable label suchas a nanoparticle, n=1 to 50 (such as n=2 to 30, n=3 to 20 or n=4 to 12)and q=1 to 10 (such as q=2 to 6 or q=3 to 4); or

wherein AHAb is an anti-hapten antibody, DL is a detectable label suchas an enzyme and n=1 to 50 (such as n=2 to 30, n=2 to 20 or n=4 to 12)and r=1 to 10 (such as r=2 to 6 or r=3 to 4).

In still other particular embodiments, a heterobifunctional PEG-linkedspecific-binding moiety-nanoparticle conjugate comprises a conjugatehaving the formula:

wherein AHAb is an anti-hapten antibody, DL is a detectable label suchas an enzyme, m=1 to 50 (such as m=2 to 30, m=3 to 20 or m=4 to 12) ands=1 to 10 (such as s=2 to 6 or s=3 to 4); or

wherein AHAb is an anti-hapten antibody, DL is a detectable label suchas a nanoparticle, m=1 to 50 (such as m=2 to 30, 2 to 20 or 4 to 12) andt=1 to 10 (such as t=2 to 6 or t=3 to 4).

In still further particular embodiments, a heterobifunctional PEG-linkedspecific-binding moiety-nanoparticle conjugate comprises a conjugatehaving the formula:

wherein AHAb is an antihapten antibody, DL is a detectable label, m=1 to50 (such as m=2 to 30, m=3 to 20 or m=4 to 12) and u=1 to 10 (such asu=2 to 6 or u=3 to 4); or

wherein SBM is a specific-binding moiety, NP is a nanoparticle, m=1 to50 (such as m=2 to 30, m=2 to 20 or m=4 to 12) and v=1 to 10 (such asv=2 to 6 or v=3 to 4).

Disclosed conjugates can be utilized for detecting one or more moleculesof interest in a biological sample in any type of assay, includingimmunohistochemical assays and in situ hybridization assays. In oneembodiment, the disclosed conjugates are used as a hapten-labeledantibody in an immunoassay, for example, a hapten-labeled primaryantibody directed to a particular molecule that is then contacted withan anti-hapten antibody conjugate including a detectable label.Alternatively, a hapten-labeled nucleic acid probe bound to a targetnucleic acid is then contacted with an anti-hapten antibody conjugateincluding a detectable label. The biological sample can be any samplecontaining biomolecules (such as proteins, nucleic acids, lipids,hormones etc.), but in particular embodiments, the biological sampleincludes a tissue section (such as obtained by biopsy) or a cytologysample (such as a Pap smear or blood smear). Other types of assays inwhich the disclosed conjugates can be used are readily apparent to thoseskilled in the art, and particular examples are discussed below.

In another aspect, a method is disclosed for preparing aspecific-binding moiety-hapten conjugate, the method including forming athiolated specific-binding moiety from a specific-binding moiety;reacting a hapten having an amine group with a maleimide/active esterbifunctional linker to form an activated hapten; and reacting thethiolated specific-binding moiety with the activated hapten to form thespecific-binding moiety-hapten conjugate.

A thiolated specific-binding moiety can be formed by reacting thespecific-binding moiety with a reducing agent to form the thiolatedspecific-binding moiety, for example, by reacting the specific-bindingmoiety with a reducing agent to form a thiolated specific-binding moietyhaving an average number of thiols per specific-binding moiety ofbetween about 1 and about 10. The average number of thiols perspecific-binding moiety can be determined by titration. Examples ofreducing agents include reducing agents selected from the groupconsisting of 2-mercaptoethanol, 2-mercaptoethylamine, DTT, DTE andTCEP, and combinations thereof. In a particular embodiment the reducingagent is selected from the group consisting of DTT and DTE, andcombinations thereof, and used at a concentration of between about 1 mMand about 40 mM.

Alternatively, forming the thiolated specific-binding moiety includesintroducing a thiol group to the specific-binding moiety. For example,the thiol group can be introduced to the specific-binding moiety byreaction with a reagent selected from the group consisting of2-Iminothiolane, SATA, SATP, SPDP, N-Acetylhomocysteinethiolactone,SAMSA, and cystamine, and combinations thereof (see, for example,Hermanson, “Bioconjugate Techniques,” Academic Press, San Diego, 1996,which is incorporated by reference herein). In a more particularembodiment, introducing the thiol group to the specific-binding moietyincludes reacting the specific-binding moiety with an oxidant (such asperiodate) to convert a sugar moiety (such as in a glycosylated portionof an antibody) of the specific-binding moiety into an aldehyde groupand then reacting the aldehyde group with cystamine. In another moreparticular embodiment, the specific binding moiety includes streptavidinand introducing the thiol group comprises reacting the streptavidin with2-iminothiolane (Traut reagent).

In other particular embodiments, reacting the hapten with amaleimide/active ester bifunctional linker to form an activatednanoparticle includes reacting the hapten with a PEG maleimide/activeester having the formula:

wherein n=1 to 50, for example, n=2 to 30 such as n=3 to 20 or n=4 to12.

In a further aspect, a method is disclosed for preparing aspecific-binding moiety-hapten conjugate composition that includesreacting a specific-binding moiety with an oxidant to form analdehyde-bearing specific-binding moiety; reacting the aldehyde-bearingspecific-binding moiety with a maleimide/hydrazide bifunctional linkerto form a thiol-reactive specific-binding moiety; and reacting thethiol-reactive specific-binding moiety with a thiolated hapten to formthe specific-binding moiety-nanoparticle conjugate. In a particularembodiment, the specific-binding moiety is an antibody and reacting thespecific-binding moiety with an oxidant to form the aldehyde-bearingspecific-binding moiety includes oxidizing (such as with periodate, I₂,Br₂, or a combination thereof, or neuramidase/galactose oxidase) aglycosylated region of the antibody to form the aldehyde-bearingantibody. In a more particular embodiment, reacting an antibody with anoxidant to form an aldehyde-bearing antibody includes introducing anaverage of between about 1 and about 10 aldehyde groups per antibody. Ina more particular embodiment, the maleimide/hydrazide bifunctionallinker has the formula:

wherein m=1 to 50, for example, m=2 to 30 such as m=3 to 20 or m=4 to12. A thiolated hapten can be formed from a hapten by introducing athiol group to the hapten (for example, by reacting a hapten with areagent selected from the group consisting of 2-Iminothiolane, SATA,SATP, SPDP, N-Acetylhomocysteinethiolactone, SAMSA, and cystamine, andcombinations thereof).

In other embodiments, a hapten-linker conjugate having a hydrazidereactive group is reacted with a carbonyl group of an aldehyde formed onan antibody to form a hapten-linker-antibody conjugate. Hapten-linkerconjugates having hydrazide reactive group are discussed further below.

4. Commercially Available Linkers

Additional linkers also are commercially available. Pierce BiotechnologyInc., of Rockford, Ill., provides certain linkers that are useful forpracticing the present invention. For example, Pierce providessulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC).Pierce also sells a sulfonamidyl compound without the sulfo group,referred to as SMCC, which also is a useful linker Sulfo-SMCC is a watersoluble, non-cleavable membrane, impermeable cross-linker. NH esters ofthis compound can react readily with primary amines at pH 7-9 to formstable amide bonds. Malemides react with sulfydryl groups at pH betweenabout 6 to 8, more typically from about 6.5 to about 7.5, to form stablethioether bonds. For use in coupling the haptens to a carrier comprisinga free amine, the sulfo-SMCC can react with the free amine of thecarrier to provide a malemide-activated carrier. The carrier typecompound then can be reacted with a hapten, such as a hapten having afree sulfidryl group or hydroxyl group, to form a conjugate according tothe present invention.

Pierce also provides additional exemplary linkers, as well as additionalinformation concerning the length for each potentially suitable forpractical use. For example, for functional groups reactive with amines,Pierce provides the following compounds: EGS (ethylene glycolbis[succinimidylsuccinate]); Sulfo-EGS (ethylene glycolbis[sulfosuccinimidylsuccinate]); DTSSP(3,3′-dithiobis[sulfosuccinimidylpropionate]); DSS (disuccinimidylsuberate); BS (bis[sulfosuccinimidyl] suberate); DSG (disuccinimidylglutarate); and MSA (methyl N-succinimidyl adipate. Examples ofsulfhydryl reactive linkers include DPDPB(1,4-di-[3′-(2′-pyridyldithio)-propionamido]butane);BM[PEO]₃(1,11-bis-maleimidotriethyleneglycol); BMH(bis-maleimidohexane); BM[PEO]₂(1,8-bis-maleimidodiethyleneglycol); HBVS(1,6-hexane-bis-vinylsulfone); DTME (dithio-bis-maleimidoethane); BMDB(1,4-bis-maleimidyl-2,3-dihydroxybutane); BMB (1,4-bis-maleimidiobutane;and BMOE (bis-maleimidoethane). Photoreactive compounds also areavailable from Pierce, including BASED(bis-[b-(4-azidosalicylamido)ethyl]disulfide) and APG (p-azidophenylglyoxal monohydrate).

5. Carbodiimide Coupling

Carbodiimides [R—N═C═N—R₁] can be used to couple haptens directly totarget molecules, including amino acids, proteins, nucleotides, andoligonucleotides. See, for example, New Biotinylating Reagent UtilizingCarbodiimide Function, Nucleic Acid Symposiums Series, No. 34, 69-70(1995), which is incorporated herein by reference. Alternatively,carbodiimide functionalities can be incorporated into or coupled to alinker as discussed above for forming hapten-linker conjugates. Ageneral synthetic scheme for making hapten-dPEGx-carbodiimides isprovided below in the working examples. Using this general syntheticscheme, several working embodiments of hapten-carbodiimides have beenmade, including nitropyrazole-dPEGx-carbodiimide,benzofurazan-dPEGx-carbodiimide, dinitrophenyl-dPEGx-carbodiimide,thiazolesulfonamide-dPEGx-carbodiimide and rotenoid-dPEGx-carbodiimide.

IV. Hapten-Linker Conjugates

Compounds of the present invention, also referred to as conjugates,typically comprise a hapten typically coupled to a linker. The haptenconjugate also may include a carrier, such as a polypeptide, protein,mononucleotide, dinucleotide, trinucleotide, oligonucleotide, or nucleicacid(s), either coupled directly to the hapten, or coupled to linkerParticular examples of carriers include immunogenic carriers, antibodiesand nucleic acid probes. The hapten, carrier and/or linker may includeone or more functional groups or moieties, typically electrophiles andelectrophile/nucleophile pairs that are useful for coupling a hapten toa carrier, either directly or indirectly through a linker. Thus, a firstgeneral formula describing certain embodiments of the present disclosureis hapten-carrier. Such compounds also optionally, and most typically,include a linker. Embodiments having a linker satisfy the formulahapten-linker-carrier. A combined formula therefore is(hapten)_(k)-(linker)_(m)-carrier_(n) where k is 1, m and n are 0 or 1,and at least one of m or n is 1. A person of ordinary skill in the artwill understand that, for the general formula, k=1, m=1 or n=1 impliesno limitation on the number or structure of the hapten, the linker orthe carrier. For example, a carrier can have multiple linkers attachedand the multiple linkers can be attached to multiple haptens to providea conjugate of the combined formula. Furthermore, a linker can includeplural subunits or be formed from various subcomponents. For example,both a carrier and a hapten can include attached linkers, wherein thelinkers can then be reacted to couple the hapten and the carriertogether.

In a particular embodiment, a conjugate according to the disclosure hasthe general structure (specific-binding moiety)-linker-hapten, and moreparticularly (specific-binding moiety)-(linker-hapten)_(p) wherep=1-200, for example p=1-50 such as 1-10. In one example, the linkercomprises a PEG linker. In more particular embodiments, the specificbinding moiety is an antibody or a nucleic acid. In another example, thespecific-binding moiety is an antibody and the linker includes acarbonyl reactive group covalently linked to an aldehyde group of anoxidized sugar moiety of an Fc region of the antibody. In yet anotherexample, the specific-binding moiety is an antibody and the linkerincludes a sulfhydryl reactive group covalently linked to a thiol groupof the antibody, wherein the thiol group is generated by reducing adisulfide bond in the antibody. In still another example, the specificbinding moiety is a nucleic acid and the linker includes a carbonylreactive group covalently linked to a cytosine residue of the nucleicacid.

In another particular embodiment, a conjugate according to thedisclosure has the general structure hapten-linker-RG, wherein RG refersto a reactive group, such as a carbonyl reactive group, a thiol reactivegroup or an amine reactive group. Although typically one hapten will belinked to one linker bearing a reactive group, it is possible to havemultiple haptens attached to one linker having a reactive group, or tohave multiple linkers having reactive groups attached to one hapten.Hapten linker conjugates such as these are particularly useful forattaching a hapten to an antibody (such as discussed in the previousparagraph) and also for attaching a hapten to an immunogenic carriersuch as KLH to provide an immunogen that can be used to stimulate ananimal to produce an antibody that specifically binds to the hapten.Thus, an antibody that specifically binds to a hapten is an aspect ofthe disclosure.

In yet another aspect, a conjugate is disclosed that includes ananti-hapten antibody (such as can be produced using a disclosedimmunogen) and a detectable label (such as a quantum dot or enzyme).Thus, a general formula for this type of conjugate is (anti-haptenantibody)_(t)-(detectable label)_(s) where t and s can eachindependently be 1-100, but more typically, t=1 and s=1-10. Conjugatesof anti-hapten antibodies with detectable labels can be used inconjunction with other hapten-carrier conjugates (such as hapten labelednucleic acid probes for target genomic sequences and hapten-labeledprimary antibodies that specifically bind to target proteins) to allowmultiplexed assays of multiple targets in a single sample.

Conjugates of the present invention may be formed by coupling adisclosed exemplary linker or linkers to a disclosed hapten or haptens.Many of the haptens have plural locations to which a linker may becoupled. Suitable linker positions with respect to the general formulaeprovided for disclosed haptens are indicated below, as are generalformulae for hapten-linker conjugates. Particular hapten-linkerconjugates are provided by reference to PEG linkers, as are protocolsfor synthesizing these compounds.

1. Oxazoles and Pyrazoles

A first general class of haptens of the present invention are oxazolesand pyrazoles, most typically nitro oxazoles and nitro pyrazoles, havingthe following general chemical formula, as discussed in more detailherein.

Any one or more of the R₁-R₄ positions can be coupled to a linker. Theposition occupied by the R₂ substituent is quite suitable for couplinglinkers to this class of haptens, as indicated below, where L is alinker and RG is a reactive functional group.

Hapten-linker conjugates have been formed using PEG-based linkers. Oneexample of such a compound, 5-nitro-3-pyrazole carbamide, is shownbelow. For this and subsequent embodiments, X is from about 2 to about24, typically from about 4 to about 12.

A particular embodiment where X is 4 is provided below.

This example satisfies the formula hapten-L-RG where L is a PEG 4 (4ethylene oxy units) and the reactive group is a carboxylic acidfunctional group. The carboxylic acid functional group has beenconverted to other reactive functional groups in working embodiments.For example, the carboxylic acid functional group can be converted to anactivated ester, such as an NHS ester, as shown below.

And, the activated ester can be converted to other useful reactivefunctional group, such as a hydrazide, as illustrated below.

2. Nitroaryl

A second general class of haptens of the present invention are nitroarylcompounds having the following general chemical formula.

Such compounds have at least one, and optionally plural, nitro groups sothat at least one of R₁-R₆ is nitro. Any of the R₁-R₆ positions notcoupled to a nitro group is a potential position for coupling linkers tothe aryl ring. Mononitrophenyl compounds are represented bynitrocinnamide hapten conjugates as illustrated below.

Working embodiments also are exemplified by 2,4-dinitrophenyl compounds.Exemplary hapten conjugates of this class are illustrated below, whereR₁-R₃ are as stated above.

Hapten-linker conjugates have been formed using PEG-based linkers. Oneexample of such a compound is shown below.

A particular embodiment had the following structure.

This example therefore satisfies the formula hapten-L-RG where L is aPEG 4 (4 ethylene oxy units) and the reactive group is a carboxylic acidfunctional group. The carboxylic acid functional group has beenconverted to other reactive functional groups in working embodiments.For example, the carboxylic acid functional group can be converted to anactivated ester, such as an NHS ester, as shown below.

The activated ester can be converted to other useful reactive functionalgroup, such as a hydrazide, as illustrated below.

3. Benzofurazan and Related Compounds

Benzofurazans and derivatives thereof are in another class of haptens ofthe present invention. A general formula for the benzofurazan-typecompounds is provided below.

The R₁-R₄ and Y substituents are as stated above. At least one of theR₁-R₄ substituents is bonded to a linker, to a carrier, or is afunctional group suitable for coupling to a linker or a carrier. The R₂and R₃ positions are most likely used to couple the linker to this classof haptens (R₂ and R₃ may be substantially identical in terms ofreactivity, particularly if R₁ and R₄ are the same). Such haptenconjugates are exemplified by the general formula provided below.

Hapten-linker conjugates have been formed using PEG-based linkers. Oneexample of such a compound, 2,1,3-benzoxadiazole-5-carbamide, is shownbelow.

A particular embodiment had the following formula.

This example satisfies the formula hapten-L-RG where L is a PEG 4 (4ethylene oxy units) and the reactive group is a carboxylic acidfunctional group. The carboxylic acid functional group has beenconverted to other reactive functional groups in working embodiments.For example, the carboxylic acid functional group can be converted to anactivated ester, such as an NHS ester, as shown below.

The activated ester can be converted to other useful reactive functionalgroup, such as a hydrazide, as illustrated below.

4. Triterpenes

Triterpenes are another class of haptens within the scope of the presentinvention. The basic ring structure common to the triterpenes has foursix-membered fused rings, A-D, as indicated below, where the R₁-R₂₁ andY substituents are as stated above.

Disclosed embodiments of triterpenes exemplifying this class of haptensalso may include an E ring, that can be of various ring sizes. Forexample, the E ring might be a 5- or 6-membered ring. Quite often, thesecompounds include an alpha-beta unsaturated ketone, such as illustratedbelow for the C ring.

A person of ordinary skill in the art will appreciate that many of thepositions occupied by the R groups in these general formulae may beuseful for coupling the haptens to a linker to form a reactiveconjugate. With reference to the alpha-beta unsaturated compounds,particular linker positions are indicated below using arrows.

For example, hapten conjugates of the present invention with particularreactive conjugate according to this class have the following formula.

Other exemplary triterpene structures and notential linker counlin2positions are provided below.

Betulinic, Ursolic and Echinocystic Acid Core Structures

Cucurbitacin Core

Hapten-linker conjugates have been formed using PEG-based linkers. Oneexample of such a compound is shown below.

This example therefore satisfies the formula hapten-L-RG where L is aPEG 4 (4 ethylene oxy units) and the reactive group is a carboxylic acidfunctional group. The carboxylic acid functional group can be convertedto an activated ester, such as an NHS ester, as shown below.

The illustrated activated ester has been coupled directly to a proteincarrier. Alternatively, the activated ester could be converted into adifferent reactive functional group, such as a hydrazide by treatmentwith a protected, e.g. a BOC-protected hydrazine reagent, if desired.

5. Ureas and Thioureas

Ureas/thioureas, particularly aryl and heteroaryl ureas and thioureas,is another class of haptens within the scope of the present invention.Aryl derivatives typically have the following formula.

At least one of the R₃-R₇ substituents also may be bonded to a linker,to a carrier, or is a functional group suitable for coupling to a linkerand/or to a carrier molecule. Alternatively, the urea/thioureafunctional group can be used to couple a linker to this class ofdisclosed haptens. An exemplary hapten conjugate, with particularreference to thioureas, is provided below.

Hapten-linker conjugates have been formed using PEG-based linkers. Oneexample of such a compound is shown below.

This example therefore satisfies the formula hapten-L-RG where L is aPEG 8 (8 ethylene oxy units) and the reactive group is a carboxylic acidfunctional group. The carboxylic acid functional group can be convertedto an activated ester, such as an NHS ester, as shown below.

The activated ester can be converted to other useful reactive functionalgroup, such as a hydrazide, as illustrated below.

Rhodamine thiourea hapten conjugates according to the present inventiontypically have the following formula.

With reference to this formula, R typically is independently selectedfrom hydrogen, aliphatic, particularly alkyl, heteroaliphatic,substituted aliphatic, such as alkyl halide, aryl, heteroaryl, andcombinations thereof. R₁ typically is independently selected fromhydrogen, aliphatic, particularly alkyl, heteroaliphatic, substitutedaliphatic, such as alkyl halide, alcohol, amine, substituted amine, suchas lower alkyl amine, one example being diethyl amine, aryl, heteroaryl,halogen, hydroxyl, and combinations thereof. R₂ typically isindependently selected from hydrogen, aliphatic, particularly alkyl,heteroaliphatic, substituted aliphatic, such as alkyl halide, alcohol,amine, substituted amine, aryl, heteroaryl, halogen, hydroxyl, andcombinations thereof. Y is oxygen, nitrogen or sulfur. A particularembodiment of a rhodamine B thiourea had the following formula.

6. Rotenone and Rotenone-based Haptens

Rotenone, and rotenone-based haptens, define another class of haptenswithin the scope of the present invention. General formulas forrotenone, and rotenone-based haptens, are provided below.

Any of the R₁-R₁₄ positions can be used to couple linkers to this classof haptens. Certain particular compounds of Formula 1 have R₆ and R₇form a double bond, such as a double bond to oxygen to form a carbonylor a double bond to a nitrogen to form an imine. Specific exemplarylinker coupling positions with reference to these hapten conjugates isprovided below.

A person of ordinary skill in the art will appreciate that the carbonylcompound can be used to bond to the linker. This class of exemplaryhapten conjugates is exemplified by the general formulas provided below.

the general formulas provided below.

One example of a hapten-linker conjugate having a PEG-based linker isshown below.

A particular embodiment had the following formula.

This example satisfies the formula hapten-L-RG where L is a PEG 8 (8ethylene oxy units) and the reactive group is a carboxylic acidfunctional group. The carboxylic acid functional group can be convertedinto a different reactive functional group, as desired, such as anactivated ester, including the NHS ester shown below.

The activated ester can be converted to other useful reactive functionalgroup, such as a hydrazide, as illustrated below.

For rotenone isoxazolines, exemplary hapten-linker conjugates had thefollowing formula.

7. Oxazole and Thiazole Sulfonamides

Oxazole and thiazole sulfonamides provide another class of haptenswithin the scope of the present invention. A general formula for oxazoleand thiazole sulfonamides is provided below.

Any one or more of the R₁-R₃ positions can be used to couple a linker orcarrier to this class of haptens to form hapten conjugates. For certainexemplary working embodiments, R₁ has been amido, such as the amidederivatives shown below. For these compounds, the R₂ and R₃ positionsare suitable for coupling to a linker. R₂, for certain workingembodiments, has been —SO₂, and has been used to couple linkers byforming a sulfonamide. Thus, a second general formula for workingembodiments of haptens exemplifying this class of haptens is indicatedbelow.

Exemplary hapten conjugates based on this general formula include thosehaving the following formula.

Hapten-linker conjugates have been formed using PEG-based linkers. Oneexample of such a compound, 2-acetamido-4-methyl-5-thiazolesulfonamide,is shown below.

A particular embodiment had the following structure.

This example satisfies the formula hapten-L-RG where L is a PEG 8 (8ethylene oxy units) and the reactive group is a carboxylic acidfunctional group. The carboxylic acid functional group can be convertedinto a different reactive functional group, as desired, such as anactivated ester, including the NHS ester shown below.

The activated ester can be converted to other useful reactive functionalgroup, such as a hydrazide, as illustrated below.

8. Coumarins

Coumarin and coumarin derivatives provide another class of haptenswithin the scope of the present invention. A general formula forcoumarin and coumarin derivatives is provided below.

Any of the R₁-R₆ positions also typically is bonded to a linker, to acarrier or is a functional group suitable for coupling to a linker or acarrier molecule. Certain working embodiments have used the positionindicated as having an R₅ substituent for coupling to linkers. Theposition occupied by the R₆ substituent can be important if fluorescenceis used to detect these compounds. Substituents other than hydrogen atthe position occupied by R₆ in the general formula are believed toquench fluorescence, although such derivatives still may bechromophores. Exemplary hapten conjugates made using this formula havethe following general formula.

Working embodiments typically were fused A-D ring systems, as indicatedbelow.

Hapten conjugates exemplifying this class are provided below.

These examples satisfy the formula hapten-L-RG where L is a PEG linkerand the reactive group is a carboxylic acid functional group. Thecarboxylic acid functional group can be converted into a differentreactive functional group, as desired, such as an activated ester,including the NHS esters shown below.

The NHS esters can be converted to other useful reactive functionalgroup, such as a hydrazide, as illustrated below.

9. Cyclolignans

Cyclolignans provide another class of haptens within the scope of thepresent invention. A first general formula, discussed herein in detail,is provided below.

At least one of the R₁-R₁₂ substituents typically is bonded to a linkeror to carrier, or is a reactive functional group capable of reactingwith a linker or carrier. At least one of R₁₂ and R₁₁ also often is anaryl group, such as a benzene ring or a substituted benzene ring.Exemplary compounds where at least one of R₁₁ and R₁₂ is an aryl grouptypically have the following general formula, where the R substituentsare as stated above.

R₉ often is -L-RG, and R₁₆-R₂₀ independently are hydrogen and alkoxy,typically lower alkoxy, such as methoxy, as shown below. The followinggeneral molecular formula indicates likely positions for couplinglinkers to this class of haptens.

Another general formula useful for describing species of compoundswithin this class is as follows.

As with all hapten conjugates of the present invention, at least one ofthe R substituents typically is bonded to a linker, is a reactivefunctional group capable of reacting with a linker, or is -L-RG. Forexample, R₉ often is -L-RG, as indicted below.

Hapten-L-RG conjugates exemplifying this class are provided below.

These examples satisfy the formula hapten-L-RG where L is a PEG linkerand the reactive group is a carboxylic acid functional group. Thecarboxylic acid functional group can be converted into a differentreactive functional group, as desired, such as an activated ester,including the NHS esters shown below.

The NHS esters can be converted to other useful reactive functionalgroups, such as a hydrazide, as illustrated below.

10. Heterobiaryl Hapten Conjugates

Heterobiaryl hapten conjugates provide another class of haptens withinthe scope of the present invention. This general class of haptens has afirst general chemical formula as below.

With reference to this general formula, A-D are selected from carbon,nitrogen, oxygen, and sulfur, and most typically are carbon or nitrogen.

At least one of the R₁-R₂ substituents typically is bonded to a linkeror to carrier, or is a reactive functional group capable of reactingwith a linker or carrier.

A particular example of a monoheteroatombiaryl hapten conjugate isillustrated below. R typically is hydroxyl or carboxyl. For hydroxylconjugates, the hydroxyl group can be converted to a halide, andsubsequently displaced using an aminocarbodiimide to produce acarbodiimide compound suitable for directly labeling biomolecules. Thecarboxyl group can be activated, such as by formation of the acid halideor NHS ester for further reaction, such as with a protected hydrazide.The synthesis of these compounds is described in further detail below.

Another general class of haptens of the present invention isheterobicyclic/biaryl compounds, typically phenyl quinolines andquinoxalines, having a first general chemical formula as below.

R₁-R₂ substituents are as stated above for this class of haptens. Aparticular example of a diheteroatombiaryl hapten conjugate isillustrated below.

As with the monoheteroatom derivatives, R typically is hydroxyl orcarboxyl. These functional groups can be used to form additionalconjugates as disclosed herein. R typically is hydroxyl or carboxyl. Forhydroxyl conjugates, the hydroxyl group can be converted to a halide,and subsequently displaced using an aminocarbodiimide to produce acarbodiimide compound suitable for directly labeling biomolecules. Thecarboxyl group can be activated, such as by formation of the acid halideor NHS ester for further reaction, such as with a protected hydrazide.The synthesis of these compounds is described in further detail below.

11. Azoaryl Conjugates

Certain disclosed embodiments of azoaryl hapten conjugates had a formulaas provided below.

R₂-R₄ are as stated above. The linker or carrier may be, for example,coupled to the azoaryl hapten by reaction with a sulfonyl halide. Oneembodiment of such a conjugate,4-(dimethylamino)azobenzene-4′-sulfonamide, has the formula providedbelow.

12. Benzodiazepine Conjugates

Particular embodiments of the benzodiazepine haptens have R₁ aryl, asindicated below, where a linker or carrier is coupled to the aryl group.For example, benzodiazepine hapten linker conjugates have a formula asindicated below.

R₂-R₅ are as stated above. More typically such substituents areindependently selected from aliphatic, particular alkyl, hydrogen andhydroxyl. Certain disclosed embodiments are phenyl compounds, asillustrated below.

Again, R₂-R₆ are as stated above. A particular embodiment,(E)-2-(2(2-oxo-2,3-dihydro-1H-benxo[b][1,4]diazepin-4yl)phenoxy)acetamide,is provided below.

R typically is hydroxyl or carboxyl. These functional groups can be usedto form additional conjugates as disclosed herein. R typically ishydroxyl or carboxyl. For hydroxyl conjugates, the hydroxyl group can beconverted to a halide, and subsequently displaced using anaminocarbodiimide to produce a carbodiimide compound suitable fordirectly labeling biomolecules. The carboxyl group can be activated,such as by formation of the acid halide or NHS ester for furtherreaction, such as with a protected hydrazide. The synthesis of thesecompounds is described in further detail below.

V. Carriers

A person of ordinary skill in the art will recognize that the haptenlinker conjugates of the present application also may include a carrierthat is coupled to the hapten, or hapten-linker, by a suitablefunctional group. Carriers can be, by way of example, and withoutlimitation, amino acids, polypeptides, proteins, and portions thereof;nucleosides, nucleotides, nucleotide chains, nucleic acids, DNA, RNA,mRNA, etc.; and combinations thereof. Typically carrier molecules areproteins, DNA, RNA, or combinations thereof.

Suitable carriers also are disclosed in published patent documents. Forexample, Waggoner et al., U.S. patent application number 2004/0057958,which is incorporated herein by reference, discloses additional suitablecarriers. Carriers may be used to enhance the immunogenicity of ahapten, or any other antigenic compound that is immunogenic,non-immunogenic, or weakly immunogenic when not associated with thecarrier. Certain properties of potential carriers also can be consideredwhen selecting a particular carrier, such as physiochemical qualitiesincluding being non-immunogenic, non-allergenic, non-antigenic, beingmetabolizable, molecular weight, solubility, particularly in aqueousphysiological solutions, such as phosphate buffered saline, for example,and capable of being conjugated (e.g., covalently bound) or associated(e.g., admixed with or associated through charge-charge interactions)with the antigenic compound.

A single carrier can be used, as well as mixtures of different carriers.Different carriers include, for example, polymers of different lengths,such, as, for example, two or more different length homopolymers, aswell as mixtures of two or more different carriers or polymers of theinvention. Using a single carrier requires producing only onecarrier-hapten complex, whereas using multiple carriers obviously ismore difficult. Using more than one carrier may be advantageous if theimmune response generated against a particular hapten or epitope varies,such as in magnitude or specificity, for example, depending upon theparticular carrier used, and the most optimal carrier is not known orhas not yet been experimentally determined.

The carrier may be a synthetic or natural polymer, substantiallyantigenic, substantially non-antigenic or biodegradable, or both.Examples of suitable polymers useful as carriers include, but are notlimited to, poly(diene), a poly(alkene), a poly(acrylic), apoly(methacrylic), a poly(vinyl ether), a poly(vinyl alcohol), apoly(vinyl ketone), a poly(vinyl halide), a poly(vinyl nitrile), apoly(vinyl ester), a poly(styrene), a poly(carbonate), a poly(ester), apoly(orthoester), a poly(esteramide), a poly(anhydride), apoly(urethane), a poly(amide), a cellulose ether, a cellulose ester, apoly(saccharide), poly(lactide-co-glycolide), a poly(lactide), apoly(glycolide), a copolyoxalate, a polycaprolactone, apoly(lactide-co-caprolactone), a poly(esteramide), a polyorthoester, apoly(a-hydroxybutyric acid), a polyanhydride or a mixture thereof. Inanother embodiment, the polymers may be a polymer or oligomer derivedfrom the polymerization or oligomerization of at least one monomerselected from an alpha hydroxycarboxylic acid or acids (such as alphahydroxycarboxylic acid comprises glycolic acid, lactic acid, a-hydroxybutyric acid, a-hydroxyisobutyric acid, a-hydroxyvaleric acid,a-hydroxyisovaleric acid, a-hydroxy caproic acid,a-hydroxy-a-ethylbutyric acid, a-hydroxyisocaproic acid,a-hydroxy-3-methylvaleric acid, a-hydroxyheptanoic acid,a-hydroxyoctanoic acid, a-hydroxydecanoic acid, a-hydroxymysristic acid,a-hydroxystearic acid, a-hydroxyligoceric acid), a lactone(3-propiolactone, tetramethyleneglycolide, b-butyrolactone,4-butyrolactone, pivalactone), a diene, an alkene, an acrylate, amethacrylate, a vinyl ether, a vinyl alcohol, a vinyl ketone, a vinylhalide, a vinyl nitrile, a vinyl ester, styrene, a carbonate, an ester,an orthoester, an esteramide, an anhydride, a urethane, an amide, acellulose ether, a cellulose ester, a saccharide, an alphahydroxycarboxylic acid, a lactone, an esteramide, or a mixture thereof.

The polymer may be derived from one or more amino acids, including bothhomopolymers and heteropolymers thereof. For example, polyglutamatederived from L-glumatic acid, D-glumatic acid or mixtures, e.g.,racemates, of these L and D isomers are used. L and/or D glutanyl,aspartly, glycyl, seryl, threonyl, and cysteinyl are all examples ofamino acids that may be used. The polymer also may be a block, graft orrandom copolymer. These include, for example, copolymers containing atleast one other amino acid, such as aspartic acid, serine, tyrosine,glycine, ethylene glycol, ethylene oxide, (or an oligomer or polymer ofany of these) or polyvinyl alcohol. Glutamic acid may, of course, carryone or more substituents and the polymers include those in which aproportion or all of the glutamic acid monomers are substituted.Particular polymer examples include, but are not limited to,poly(l-glutamic acid), poly(d-glutamic acid), poly(dl-glutamic acid),poly(l-aspartic acid), poly(d-aspartic acid), poly(dl-aspartic acid),poly(l-serine), poly(d-serine), poly(dl-serine), poly(l-tyrosine),poly(d-tyrosine), poly(dl-tyrosine), poly(l-glysine), poly(d-glysine),poly(dl-glysine), poly(l-threonine), poly(d-threonine),poly(dl-threonine), poly(d-cysteine), poly(l-cysteine), andpoly(dl-cysteine). In further embodiments, the polymers are copolymers,such as block, graft or random copolymers, of the above listedpoly(amino acids) with polyethylene glycol, polycaprolactone,polyglycolic acid and polylactic acid, as well as poly(2-hydroxyethyl1-glutamine), chitosan, carboxymethyl dextran, hyaluronic acid, humanserum albumin and alginic acid, with poly-glutamic acids beingparticularly preferred.

The molecular weight of suitable polymers may vary. Typically, however,the molecular weight is from about 1,000 kilodaltons molecular weight toless than 10,000,000 kilodaltons.

Working embodiments exemplifying protein carriers included bovinethyroglobulin, keyhole limpet hemocyanin, or bovine serum albumin.

Hapten conjugates of the present application include a reactivefunctional groups for coupling the hapten to a carrier, or the hapten toa linker to form a hapten-linker compound. For protein coupling,proteins include various functional groups, typically nucleophiles, thatcan be coupled to suitable electrophilic functional groups to formhapten conjugates. For example, a free amine (—NH₂) or secondary aminecan be used to couple the protein to the hapten or hapten-linkercompound to form amides by reaction with carboxylic acids or carboxylicacid derivatives, such as acid halides (—CO), succinimide ester, etc.;alkyl halides can be used to form amines; carbonyl compounds, such asketones and aldehydes, can be used to form imines; and combinationsthereof. Certain amino acids include other reactive functional groupssuitable for coupling carriers of haptens, including reactive hydroxyland/or sulfhydryl groups. Exemplary couplings include ester and lactoneformation by reaction with a carboxylic acid or carboxylic acidderivative; ether formation; and combinations thereof.

VI. Signal Generating Moieties

Conjugates comprising signal generating moieties, such as conjugates ofspecific-binding moieties and signal-generating moieties, can be used inassays for detecting specific target molecules in biological samples.The signal-generating portion is utilized to provide a detectable signalthat indicates the presence/and or location of the target. Examples ofsignal-generating moieties include, by way of example and withoutlimitation: enzymes, such as horseradish peroxidase, alkalinephosphatase, acid phosphatase, glucose oxidase, β-galactosidase,β-glucuronidase or β-lactamase. Horseradish peroxidase is widely used asa label for immunoglobulins in many different immunochemistryapplications including ELISA, immunoblotting and immunohistochemistry.In addition to other possible disclosed embodiments, HRP can beconjugated to antibodies by several different methods includingglutaraldehyde, periodate oxidation, through disulfide bonds, and alsovia amino and thiol directed cross-linkers. HRP is the smallest and moststable of the three most popular enzyme labels (HRP, alkalinephosphatase, and B-galactosidase) and its glycosylation leads to lowernon-specific binding; fluorescent molecules (such as fluoresceins,coumarins, BODIPY dyes, resorufins, rhodamines; additional examples canbe found in The Handbook—A Guide to Fluorescent Probes and LabelingTechnologies, Invitrogen Corporation, Eugene, Oreg.), detectableconstructs (such as fluorescent constructs like quantum dots, which canbe obtained, for example, from Invitrogen Corporation, Eugene, Oreg.;see, for example, U.S. Pat. Nos. 6,815,064, 6,682,596 and 6,649,138,each of which patents is incorporated by reference herein), metalchelates (such as DOTA and DPTA chelates of radioactive or paramagneticmetal ions like Gd³⁺) and liposomes (such as liposomes sequesteringfluorescent molecules.

When the signal-generating moiety includes an enzyme, a chromageniccompound, fluorogenic compound, or luminogenic compound can be used togenerate a detectable signal (a wide variety of such compounds areavailable, for example, from Invitrogen, Eugene Oreg.). Particularexamples of chromogenic compounds include di-aminobenzidine (DAB),4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate(BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red, AP Orange, APblue, tetramethylbenzidine (TMB), 2,2′-azino-di-[3-ethylbenzothiazolinesulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN),nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD),5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal),methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitorphenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blueand tetrazolium violet.

Labeled secondary antibodies can be purchased from a number of sources,such as, but not limited to, Pierce Co. Amersham and EvidentTechnologies provide a broad range of conjugated antibody possibilities.CyDye, EviTag Quantum Dot, fluorescein (FITC), and rhodamine can beutilized. These conjugates span a variety of applications, colors, andemission ranges. The EviTag Quantum Dots from Evident Technologies offerphoto-stability and multicolor fluorescence in a variety of wavelengths,with the advantage over organic fluorophores of improved photostability,color multiplexing, and single source excitation. Each Evitag generatesa sharp emission wavelength making them ideal for multiplexing in intactcell environments.

The Amersham CyDyes offer superior photostability over a broad range ofpH values. For a tutorial on fluorescent markers, with the chemicalstructures of the labels, see: http://www.hmds.org.uk/fluorochrome.html.See the following link on how to label with haptens:http://probes.invitrogen.com/handbook/boxes/2020.html

One type of detectable conjugate is a covalent conjugate of an antibodyand a fluorophore. Directing photons toward the conjugate that are of awavelength absorbed by the fluorophore stimulates fluorescence that canbe detected and used to qualitate, quantitate and/or locate theantibody. A majority of the fluorescent moieties used as fluorophoresare organic molecules having conjugated pi-electron systems. While suchorganic fluorophores can provide intense fluorescence signals, theyexhibit a number of properties that limit their effectiveness,especially in multiplex assays and when archival test results areneeded.

Organic fluorophores can be photo-bleached by prolonged illuminationwith an excitation source, which limits the time period during whichmaximal and/or detectable signals can be retrieved from a sample.Prolonged illumination and/or prolonged exposure to oxygen canpermanently convert organic fluorophores into non-fluorescent molecules.Thus, fluorescence detection has not been routinely used when anarchival sample is needed.

Chromophoric and/or fluorescent semiconductor nanocrystals, also oftenreferred to as quantum dots, can be used for identifying complexes.Nanocrystalline quantum dots are semiconductor nanocrystallineparticles, and without limiting the present invention to use withparticle light emitters of a particular size, typically measure 2-10 nmin size (roughly the size of typical proteins). Quantum dots typicallyare stable fluorophores, often are resistant to photo bleaching, andhave a wide range of excitation, wave-length and narrow emissionspectrum. Quantum dots having particular emission characteristics, suchas emissions at particular wave-lengths, can be selected such thatplural different quantum dots having plural different emissioncharacteristics can be used to identify plural different targets.Quantum dot bioconjugates are characterized by quantum yields comparableto the brightest traditional dyes available. Additionally, these quantumdot-based fluorophores absorb 10-1000 times more light than traditionaldyes. Emission from the quantum dots is narrow and symmetric, whichmeans overlap with other colors is minimized, resulting in minimal bleedthrough into adjacent detection channels and attenuated crosstalk, inspite of the fact that many more colors can be used simultaneously.Symmetrical and tuneable emission spectra can be varied according to thesize and material composition of the particles, which allows flexibleand close spacing of different quantum dots without substantial spectraloverlap. In addition, their absorption spectra are broad, which makes itpossible to excite all quantum dot color variants simultaneously using asingle excitation wavelength, thereby minimizing sampleautofluorescence.

Furthermore, it has been found that pegylation, the introduction ofpolyethylene glycol groups onto the quantum dot conduits, cansubstantially decrease non-specific protein:quantum dot interaction.Certain quantum dots are commercially available, such as from QuantumDot Corp., of Hayward, Calif., and Invitrogen.

Standard fluorescence microscopes are an inexpensive tool for thedetection of quantum dot bioconjugates. Since quantum dot conjugates arevirtually photo-stable, time can be taken with the microscope to findregions of interest and adequately focus on the samples. Quantum dotconjugates are useful any time bright photo-stable emission is requiredand are particularly useful in multicolor applications where only oneexcitation source/filter is available and minimal crosstalk among thecolors is required. For example, quantum dots have been used to formconjugates of Streptavidin and IgG to label cell surface markers andnuclear antigens and to stain microtubules and actin (Wu, X. et al.(2003). Nature Biotech. 21, 41-46).

As an example, fluorescence can be measured with the multispectralimaging system Nuance™ (Cambridge Research & Instrumentation, Woburn,Mass.). As another example, fluorescence can be measured with thespectral imaging system SpectrView™ (Applied Spectral Imaging, Vista,Calif.). Multispectral imaging is a technique in which spectroscopicinformation at each pixel of an image is gathered and the resulting dataanalyzed with spectral image-processing software. For example, theNuance system can take a series of images at different wavelengths thatare electronically and continuously selectable and then utilized with ananalysis program designed for handling such data. The Nuance system isable to obtain quantitative information from multiple dyessimultaneously, even when the spectra of the dyes are highly overlappingor when they are co-localized, or occurring at the same point in thesample, provided that the spectral curves are different. Many biologicalmaterials autofluoresce, or emit lower-energy light when excited byhigher-energy light. This signal can result in lower contrast images anddata. High-sensitivity cameras without multispectral imaging capabilityonly increase the autofluorescence signal along with the fluorescencesignal. Multispectral imaging can unmix, or separate out,autofluorescence from tissue and, thereby, increase the achievablesignal-to-noise ratio.

Haptens can be conjugated to quantum dots, and quantum dot fluorescencecan be stimulated, such as by using fluorescence resonance energytransfer (FRET) whereby low-wavelength light stimulates quantum dotfluorescence. Invitrogen has determined that biotin-conjugated quantumdots had a 100-fold lower limit of detection for the biotin derivativebiocytin than anti-biotin Alexa Fluor. Fully biotinylated quantum dotswere 10-fold less sensitive than quantum dots with 25 percent biotincoverage.

Quantum dot use has so far been limited by their lack ofbiocompatibility. New advances in surface coating chemistry, however,have helped to overcome these problems. See, for example, Wu, X. et al.Immunofluorescent labeling of cancer marker Her2 and other cellulartargets with semiconductor quantum dots, Nature Biotechnol. 21, 41-46(2003); Jaiswal, J. K., Mattoussi, H., Mauro, J. M. & Simon, S. M.Long-term multiple color imaging of live cells using quantum dotbioconjugates, Nature Biotechnol. 21, 47-51 (2003); and Dubertret, B. etal. In vivo imaging of quantum dots encapsulated in phospholipidmicelles. Science 298, 1759-1762 (2002).

Quantum dots also have been conjugated to biorecognition molecules, Id.,such as streptavidin. These conjugates have been used on both fixedcells and tissue sections. In addition, cell-surface proteins and theendocytic compartments of live cells have been labelled with quantum dotbioconjugates.

Fluorescent proteins also can be used as a carrier, or can be coupled toa carrier, to facilitate visualization. For example, green fluorescentprotein (GFP) was originally isolated from the light-emitting organ ofthe jellyfish Aequorea victoria. Chimeric GFP fusions can be expressedin situ by gene transfer into cells, and can be localized to particularsites within the cell by appropriate targeting signals. Spectralvariants with blue, cyan and yellowish-green emissions have beensuccessfully generated from the Aequorea GFP, but none exhibit emissionmaxima longer than 529 nm. GFP-like proteins have been isolated fromAnthozoa (coral animals) that significantly expanded the range of colorsavailable for biological applications. The family of ‘GFP-like proteins’deposited in sequence databases now includes approximately 30significantly different members. Fluorescent proteins refers to proteinsthat can become spontaneously fluorescent through the autocatalyticsynthesis of a chromophore.

Proteins that fluoresce at red or far-red wavelengths (red fluorescentproteins or RFPs) are known. RFPs can be used in combination with otherfluorescent proteins that fluoresce at shorter wavelengths for bothmulticolour labelling and fluorescence resonance energy transfer (FRET)experiments. Commercially available RFPs are derived from two wild-typeGFP-like proteins. DsRed (drFP583) has excitation and emission maxima at558 nm and 583 nm, respectively. A far-red fluorescent protein wasgenerated by mutagenesis of a chromoprotein that absorbs at 571 nm.HcRed1 (Clontech) has excitation and emission maxima at 588 nm and 618nm, respectively. The fluorescent protein that emits fluorescence at thelongest wavelength (without any mutations being introduced) is eqFP611,cloned from the sea anemone Entacmaea quadricolor. This protein absorbsat 559 nm and emits at 611 nm. As many spectral variants have emerged,more investigators are becoming interested in the simultaneous imagingof multiple fluorophores and/or FRET signals.

Fusion proteins also can be used to form hapten conjugates of thepresent invention. There are at least three points to consider whencreating a functional fluorescent protein: the fluorescent protein mustfold correctly to fluoresce; the host protein must fold correctly to befunctional; and the integrity of the chimeric protein must bemaintained.

The length and sequence of any linker between the fluorescent proteinand host protein should be optimized for each specific application. Themost widely used linker designs have sequences that primarily consist ofglycine (Gly) and serine (Ser) stretches, Ser residues beinginterspersed to improve the solubility of a poly-Gly stretch.

The decision of whether to fuse a fluorescent protein to the amino orcarboxyl terminus of a protein depends on the properties of the protein.For example, a particular terminus might need to be preserved to retainproper protein function or to ensure correct localization. This decisionmight also be made on the basis of structural aspects of the particularfluorescent protein. For example, Aequorea GFP has a floppy carboxylterminal tail of approximately ten amino acids, which makes its fusionto the amino terminus of other proteins possible without the addition ofa linker. By contrast, DsRed is more successfully fused to the carboxylterminus of proteins of interest, because the amino termini projectfully from a tetrameric complex of DsRed. If neither end of a hostprotein can be modified, it is possible to insert the fluorescentprotein into the middle of the protein.

Citrine and Venus, two bright versions of a yellow-emitting mutant ofGFP (YFP) that mature efficiently, have recently been developed.

Two recently developed varieties of DsRed, known as T1 and E57, displayimproved maturation, making them preferable for use in dual-colorexperiments.

Fluorescence of some GFP variants can be ‘photoactivated’ by specificillumination, which provides the advantage that fluorescence can beturned on at a chosen time point. Three fluorescent proteins thatundergo photochemical modification in or near the chromophore have beendeveloped, PA-GFP, Kaede and KFP1, that enable selective activation offluorescence signals after specific illumination, and can be used tofluorescently mark individual cells, organelles or proteins.

Table 1 provides additional examples of signal generating moieties andconjugates comprising such moieties.

TABLE 1 Exemplary Antibody-Detectable Label Conjugates Label LabelAntibody Conjugate Emitted Excitation Emission Label Recommended for . .. Color (nm) (nm) Lake Placid Blue (EviTag ™ Quantum Dot) Flowcytometry, immunoblots, and fluorescent microscopy

<450 490 Fluorescein (i.e. FITC) Flow cytometry, incl. BD FACS systemsand Guava System, and fluorescent microscopy

 494 518 Adirondack Green (EviTag ™ Quantum Dot) Flow cytometry,immunoblots, and fluorescent microscopy

<450 520 Rhodamine Green Fluorescent microscopy

 502 527 Catskill Green (EviTag ™ Quantum Dot) Fluorescent microscopy

<450 540 Rhodamine 6G Flow cytometry, immunoblots, and fluorescentmicroscopy

 525 555 Hops Yellow (EviTag ™ Quantum Dot) Flow cytometry, immunoblots,and fluorescent microscopy

<450 560 Amersham Cy3 Fluorescent microscopy

 550 565 R-Phycoerythrin (PE) Flow cytometry, Luminex ® and Guavasystems, FRET assays, and capillary electrophoresis; use with FITC fordouble labeling

(495)565 575 Rhodamine Red Flow cytometry, fluorescent microscopy

 560 580 Birch Yellow (EviTag ™ Quantum Dot) Fluorescent microscopy

<450 580 Amersham Cy3.5 Fluorescent microscopy

 581 596 Fort Orange (EviTag ™ Quantum Dot) Flow cytometry, immunoblots,and fluorescent microscopy

<450 600 SulfoRhodamine (Alias Texas Red ®) Flow cytometry andfluorescent microscopy

 596 615 Amersham Cy5 Immunoblot, incl. Amersham Typhoon System. andimmunofluorescent applications

 650 670 Allophycocyanin (APC) FRET assays and HTRF assays

 652 670 Amersham Cy5.5 Immunoblot, especially LI-COR Odyssey systems

 675 694 Biotin Flow cytometry and other fluorescent applications

—Many of these labels can be used with multiple antibodies that do notcross-react to create custom multiplexed assays.

VII. Processes for Forming Hapten Conjugates—Reaction Schemes

The following schemes provide exemplary embodiments of a method usefulfor making conjugates of the present invention. Other syntheticmethodologies also are useful for making such conjugates, and thefollowing schemes should not be construed to limit the method to theparticular synthetic methodologies depicted.

1. Nitropyrazole Conjugates

Scheme 1 illustrates one method suitable for coupling exemplarynitropyrazole haptens to an alkylene oxide linker, and subsequently to aprotein carrier.

Scheme 1 illustrates coupling the exemplary nitropyrazole hapten is toan exemplary ethylene glycol linker via the pendent carboxylic acidfunctional group. The first step is forming an N-hydroxysuccinimide(NHS) ester from nitropyrazole. This “activates” the ester forsubsequent reaction with a nucleophile. Formation of the NHS ester wasachieved in working embodiments using dicyclohexylcarbodiimide (DCC) asa coupling reagent. Dichloromethane was used as the solvent, andtriethylamine was added as a base. The NHS ester is then ready forcoupling to a nucleophile.

A first possible synthetic path is reacting the activated ester with adiamine to produce an amide having a terminal amine. The diamine can bea protected diamine, as illustrated in Scheme 1 where the BOC-ethylenediamine compound is used. The BOC-protected amide is then deprotectedusing trifluoroacetic acid (TFA) in dichloromethane. The deprotectedcompound can then be reacted with a maleimide-PEG-NHS ester to couple alinker to the hapten. The linker also includes a reactive functionalgroup at the terminal end.

As another alternative, the hapten having an activated ester can becoupled to a linker to form an amide having either a terminal carboxylicacid functional group or a terminal hydroxyl group. A second DCCcoupling reaction can be performed to again activate the carboxylic acidfunctional group, which is reacted with the illustrated protectedhydrazine reagent. Deprotection in hydrochloric acid produced theillustrated hydrazide. Alternatively, nitropyrazole hapten-PEG linkerhaving an activated ester pendent functional group is suitable forreacting with a carrier protein to form an immunogen.

Another alternative synthetic path is illustrated using the hapten-PEGlinker having a terminal hydroxyl group. The hydroxyl terminatedcompound can be reacted with mesyl chloride, followed by reaction withiodide to provide the iodo-substituted derivative. This compound can bereacted with the illustrated dimethyl amine carbodiimide to produce acompound useful for direct labeling of a biomolecule.

2. Nitrophenyl Conjugates

Scheme 2 illustrates exemplary dinitrophenyl haptens coupled to analkylene oxide (PEG) linker. These hapten-linker conjugates subsequentlycan be derivatized as desired or directly coupled to a carrier.

The exemplary dinitrophenyl hapten was coupled to an alkylene oxidelinker, namely a bifunctional polyethylene glycol having both a pendentfree acid and amine. In a first approach, the dinitrophenyl hapten wascoupled to ethylene diamine via substitution, with the ring positionoccupied by fluorine being activated for nucleophilic substitution bythe presence of the ortho and para nitro groups. The resulting compoundincludes a terminal nucleophilic amine for coupling to amaleimide-PEG-NHS ester.

Alternatively, the dinitrophenyl hapten can be reacted with an amino-PEGcompound having either a terminal carboxlic acid or hydroxyl group. Withreference to the carboxylic acid derivative, this compound was reactedwith the dinitrophenyl hapten to substitute for fluorine. An NHS esterwas then formed using DCC in dichloromethane. The activated ester issuitable for derivatizing as desired, such as by reaction with theillustrated protected hydrazine reagent, followed by deprotection usingan acid, such as hydrochloric or trifluoracetic acid. Alternatively, theactivated ester is suitable for coupling to a carrier protein to form animmunogen.

As yet another alternative, the dinitrophenyl hapten can be reacted withan amino-PEG linker to produce a compound having a terminal hydroxylgroup. The hydroxyl terminated compound can be reacted with mesylchloride, followed by reaction with iodide to provide aniodo-substituted derivative. This compound can be reacted with theillustrated dimethyl amine carbodiimide to produce a compound useful fordirect labeling of a biomolecule.

A second example of a synthetic pathway for making cinnamide-basedconjugates is provided below in Scheme 3. The exemplary nitrophenylhapten was converted to the corresponding NHS ester using DCC. The NHSester was reacted with ethylene diamine. The resulting compound includesa terminal nucleophilic amine for coupling to a maleimide-PEG-NHS ester.

Alternatively, the nitrophenyl hapten can be reacted with an amino-PEGcompound having either a terminal carboxylic acid or hydroxyl group.With reference to the carboxylic acid derivative, this compound wasreacted with the nitrophenyl. An NHS ester was then formed using DCC indichloromethane. The activated ester is suitable for derivatizing asdesired, such as by reaction with the illustrated protected hydrazinereagent, followed by deprotection in hydrochloric acid. Alternatively,the activated ester is suitable for coupling to a carrier protein toform an immunogen.

As yet another alternative, the nitrophenyl hapten can be reacted withan amino-PEG linker to produce a compound having a terminal hydroxylgroup. The hydroxyl terminated compound can be reacted with mesylchloride, followed by reaction with iodide to provide aniodo-substituted derivative. This compound can be reacted with theillustrated dimethyl amine carbodiimide to produce a compound useful fordirect labeling of a biomolecule.

3. Benzofurazan Conjugates

Scheme 4 illustrates synthetic methodologies suitable for couplingexemplary benzofurazan haptens to an alkylene oxide linker.

With reference to Scheme 4, the exemplary benzofurazan hapten includes acarboxylic acid functional group. The first step is activation of thecarboxylic acid functional group by reaction with NHS using DCC as acoupling agent to form an activated ester, or by formation of an acidchloride. As a first option, the activated ester can be reacted with adiamine to produce a terminal amine. In certain embodiments, the diamineis a protected diamine, such as a BOC-protected diamine, as illustratedbelow in Scheme 5. Following the coupling reaction, the BOC protectinggroup can be removed in acid, such as trifluoroacetic acid. Thedeprotected compound is then reacted with a maleimide-PEG-NHS ester tocouple a linker to the hapten.

As a second alternative illustrated by Scheme 4, the activated ester isnow ready for coupling with a linker, if desired, such as the exemplarybifunctional alkylene oxide linkers, i.e. PEG linkers. Exemplary PEGlinkers may have both an amine and a carboxylic acid group or an amineand a hydroxyl group. Reaction of activated ester compound with linkerprovides either a carboxylic acid terminated compound or a hydroxylgroup terminated compound. The carboxylic acid can be converted to anactivated ester by reaction with NHS using DCC as a coupling agent. Thisactivated ester can be reacted with the illustrated protected hydrazinereagent, followed by deprotection in hydrochloric acid.

Alternatively, the hydroxyl terminated compound can be reacted withmesyl chloride, followed by reaction with iodide to provide theiodo-substituted compound. This compound can be reacted with theillustrated dimethyl amine carbodiimide to produce a compound useful fordirect labeling of a biomolecule.

Another alternative synthesis path for making a maleimide-dPEG₈conjugate is provided below in Scheme 5. The acid chloride is thenreacted with a BOC-protected hydrazide, followed by deprotection usingtrfilfuoroacetic acid. The hydrazide is then reacted withmaleimide-dPEG8-NHS to produce the illustrated conjugate.

4. Triterpene-Linker Conjugates and Triterpene Immunogens

Scheme 6 illustrates one method suitable for coupling exemplarytriterpene haptens to an alkylene oxide linker to form hapten-linkerconjugates. The hapten-linker conjugates can be further derivatized asdesired, or can be directly coupled to a protein carrier molecule.

With reference to Scheme 6, starting compound 40 was oxidized to ketone41 using pyridinium dichromate (PDC). The NHS activated ester 42 wasthen formed using DCC coupling in dichloromethane Activated ester 42 wasthen reacted with a bifunctional PEG-4 linker 43 comprising both anamine and carboxylic acid functional group to form amide 44. Thecarboxylic acid functional group of compound 44 was converted to theactivated ester 45 again using NHS and DCC. Activated ester 45 was thencoupled to an immunogenic protein carrier to form immunogen 46.

5. Urea- and Thio Urea-Based Hapten-Linker Conjugates and Immunogens

Scheme 7 illustrates one method suitable for coupling exemplary urea andthioureas-based haptens to an alkylene oxide linker, and subsequently toa protein carrier molecule.

With reference to Scheme 7, starting isothiocyanate compound 51 wasreacted with a PEG-4 linker 52 comprising both an amine and a carboxylicacid functional group to form thiourea 53. The carboxylic acidfunctional group of compound 53 was converted to the activated ester 54using NHS and DCC. Activated ester 54 was then coupled to protectedhydrazine reagent 55, followed by deprotection in 3M hydrochloric acid,to form compound 56. Alternatively, activated ester 54 can be coupled toa carrier to form immunogen 58.

6. Rotenone-Based Hapten-Linker Conjugates and Immunogens

Scheme 8 illustrates one method suitable for coupling exemplaryrotenone-based haptens to an alkylene oxide linker, and subsequently toa protein carrier molecule

Starting compound 60 was treated with NH₂OH—HCl to form an intermediateoxime, which was then reacted with alpha bromoacetic acid, compound 61,to form oxime 62. The carboxylic acid functional group of compound 62was converted into an NHS ester using DCC to form compound 63. Compound63 was then coupled to an exemplary PEG-4 linker 64, having both anamine and a carboxylic acid functional group, to produce compound 65.The carboxylic acid functional group of compound 65 was converted to theNHS ester 66 by reaction with NHS using DCC as a coupling agent.Compound 66 was then treated with BOC-protected hydrazine compound 67,and then deprotected using 3M hydrochloric acid in dioxane, to producecompound 68. A person of ordinary skill in the art will appreciate thatcompound 66 also could be coupled to a carrier, such as a proteincarrier, as disclosed herein to form an immunogen.

Scheme 9 illustrates synthetic paths used with rotenone isoxazolines.

Scheme 9 illustrates making rotenone isoxazoline conjugates bysequentially treating the starting compound with ammonium hydroxide,followed by bromoacetic acid. This results in ring rearrangement toproduce rotenone isoxazolines having a terminal carboxylic acidfunctional group. The NHS ester was produced usingN-3-dimethylaminopropyl-N′-ethylcarbodiimide (EDAC). As a first option,the NHS ester can be reacted with a diamine to produce an amide having aterminal amine. This compound can then be reacted with amaleimide-PEG-NHS ester to couple to the hapten a PEG linker having areactive terminus.

As a second alternative illustrated by Scheme 9, the NHS ester is readyfor coupling with a linker, if desired, such as the exemplarybifunctional alkylene oxide linkers, i.e. PEG linkers. These exemplaryPEG linkers have plural reactive functional groups, such as an amine anda carboxylic acid group or an amine and a hydroxyl group. Reacting theNHS ester with a linker provides either a carboxylic acid-terminatedcompound or a hydroxyl group-terminated compound. The carboxylic acidcan be converted to an NHS ester using DCC as a coupling agent. The NHSester can be reacted with the illustrated protected hydrazine reagent,followed by deprotection in hydrochloric acid, to produce theamino-terminated amide.

Alternatively, the hydroxyl terminated compound can be reacted withmesyl chloride, followed by reaction with a halide, such as iodide, toprovide the halide-substituted compound. This compound can be reactedwith the illustrated dimethyl amine carbodiimide to produce a compounduseful for direct labeling of a biomolecule.

7. Oxazole- and Thiazole-Based Conjugates

Scheme 10 illustrates one method suitable for coupling exemplaryoxazole- and thiazole-based haptens to an exemplary alkylene oxidelinker. The hapten-linker conjugate then can be derivatized as desired,or can be directly coupled to a protein carrier molecule to form animmunogen.

With reference to Scheme 10, the exemplary thiazole hapten, having areactive sulfonyl chloride functional group, was reacted with eitherethylene diamine or an exemplary bifunctional PEG₈ linker. As a firstoption, the thiazole can be reacted with a BOC-protected ethylenediamine, followed by deprotection using TFA, to produce an amide havinga terminal amine. This compound can then be reacted with amaleimide-PEG-NHS ester to couple a linker to the hapten. Alternatively,the compound can be coupled to directly to carrier protein to form animmunogen.

Alternatively, the thiazole hapten can be reacted with an amino-dPEGlinker having either a terminal hydroxyl group or a terminal carboxylicacid group. The carboxylic acid-terminated linker can be converted tothe NHS ester using DCC, followed by reaction with a BOC protectedhydrazide. The BOC group can be removed using an acid, such as 3M HCl,to form the hydrazide terminated conjugate.

The hydroxyl-terminated thiazole sulfonamide PEG conjugate can bereacted with mesyl chloride, followed by reaction with a halide, such asiodide, to provide the halide-substituted compound. This compound can bereacted with the illustrated dimethyl amine carbodiimide to produce acompound useful for direct labeling of a biomolecule.

8. Coumarin-Based Hapten-Linker Conjugates and Immunogens

Scheme 11 illustrates one method suitable for coupling exemplarycoumarin-based haptens to an exemplary alkylene oxide linker. Theresulting hapten-linker conjugate can be derivatized further as desired,or can be coupled to a carrier, such as a protein carrier, to form animmunogen.

With reference to Scheme 11, the starting compound includes a carboxylicacid functional group that was converted to an NHS using DCC as acoupling agent. As a first option, the NHS ester can be reacted withethylene diamine to produce an amide having a terminal amine. Thiscompound can then be reacted with a maleimide-PEG-NHS ester to couple tothe hapten a linker having a reactive terminal functional group.

Alternatively, the NHS ester can be coupled with the exemplarybifunctional PEG₈ to produce amides having either a terminal carboxylicacid or hydroxyl functional group. The carboxylic acid functional groupcan be converted to an NHS ester using DCC as a coupling agent. The NHSester was then reacted with the protected hydrazine compound, followedby deprotection in 3M hydrochloric acid in dioxane, to produce thehydrazide. Alternatively, the NHS ester can be coupled to an immunogenicprotein to produce an immunogen.

The hydroxyl-terminated coumarin PEG conjugate can be reacted with mesylchloride, followed by reaction with a halide, such as iodide, to providethe halide-substituted compound. This compound can be reacted with theillustrated dimethyl amine carbodiimide to produce a compound useful fordirect labeling of a biomolecule.

9. Cyclolignan-Linker Conjugates and Immunogens

Scheme 12 illustrates one method suitable for coupling exemplaryPodophyllotoxin-based haptens to an exemplary alkylene oxide linker. Thehapten-linker conjugate then can be further derivatized as desired, ordirectly coupled to a carrier molecule, such as a protein carriermolecule.

With reference to Scheme 12, the starting alcohol was oxidized to thecorresponding ketone using manganese dioxide oxidation indichloroethane. The ketone was then converted to an intermediate oxime(not shown), followed by ring rearrangement to the 5-memberedheterocycle, thereby producing a compound having a carboxylic acidfunctional group by ring opening of the lactone. This compound wasconverted to the NHS ester, using either DCC or EDAC. As a first option,the NHS ester can be reacted with ethylene diamine to produce an amidehaving a terminal amine. This compound can then be reacted with amaleimide-PEG-NHS ester to couple a linker to the hapten.

Alternatively, the NHS ester was coupled with a PEG₈ linker to producean amide having either a terminal carboxylic acid or hydroxyl functionalgroup. The carboxylic functional group of the amide was converted to theNHS ester. This compound was then coupled to a protein carrier toproduce an immunogen. Alternatively, the NHS ester was reacted with aBOC-protected hydrazine reagent, followed by deprotection in 3Mhydrochloric acid in dioxane, to produce the hydrazide.

The hydroxyl-terminated PEG conjugate can be reacted with mesylchloride, followed by reaction with a halide, such as iodide, to providethe halide-substituted compound. This compound can be reacted with theillustrated dimethyl amine carbodiimide to produce a compound useful fordirect labeling of a biomolecule.

10. Heteroaryl Conjugates

Schemes 13 and 14 illustrate one method suitable for coupling exemplaryheteroaryl-based haptens to an exemplary alkylene oxide linker. Thehapten-linker conjugate then can be further derivatized as desired, ordirectly coupled to a carrier molecule, such as a protein carriermolecule.

With reference to Schemes 13 and 14, the starting compounds each includea carboxylic acid functional group. In a first approach, the carboxylicacid can be converted to an ethyleneamino amide by reaction withBOC-protected ethylene diamine using HOBT/EDAC. The BOC protecting groupis removed using an acid, such as TFA in dichloromethane. This compoundcan then be reacted with a maleimide-PEG-NHS ester to couple a linker tothe hapten.

Alternatively, the NHS ester can be coupled to a PEG₈ linker to producean amide having either a terminal carboxylic acid or hydroxyl functionalgroup. The carboxylic functional group of the amide can be converted toan NHS ester using DCC as coupling agent. The NHS ester was reacted witha BOC-protected hydrazine reagent, followed by deprotection in 3Mhydrochloric acid in dioxane, to produce the hydrazide. Alternatively,the NHS ester can be coupled to a protein carrier to produce animmunogen.

The hydroxyl-terminated PEG conjugate can be reacted with mesylchloride, followed by reaction with a halide, such as iodide, to providethe halide-substituted compound. This compound can be reacted with theillustrated dimethyl amine carbodiimide to produce a compound useful fordirect labeling of a biomolecule.

11. Azoaryl Conjugates

Scheme 15 illustrates one method suitable for coupling exemplaryazoaryl-based haptens to an exemplary alkylene oxide linker. Thehapten-linker conjugate then can be further derivatized as desired, ordirectly coupled to a carrier molecule, such as a protein carriermolecule.

With reference to Scheme 15, the exemplary azoaryl hapten, having areactive sulfonyl chloride functional group, was reacted with eitherethylene diamine or an exemplary bifunctional PEG₈ linker. As a firstoption, the azoaryl compound can be reacted with ethylene diamine toproduce a sulfamide having a terminal amine. This compound can then bereacted with a maleimide-PEG-NHS ester to couple to the hapten a linkerhaving a reactive terminal functional group.

Alternatively, the reactive sulfonyl chloride can be reacted with a PEG₈linker to produce a sulfamide having either a terminal carboxylic acidor hydroxyl functional group. The carboxylic functional group of theamide can be converted to an NHS ester using DCC as coupling agent. TheNHS ester was reacted with a BOC-protected hydrazine reagent, followedby deprotection in 3M hydrochloric acid in dioxane, to produce thehydrazide. Alternatively, the NHS ester can be coupled to a proteincarrier to produce an immunogen.

The hydroxyl-terminated PEG conjugate can be reacted with mesylchloride, followed by reaction with a halide, such as iodide, to providethe halide-substituted compound. This compound can be reacted with theillustrated dimethyl amine carbodiimide to produce a compound useful fordirect labeling of a biomolecule.

12. Benzodiazepine Conjugates

Scheme 16 illustrates one method suitable for coupling exemplarybenzodiazepine-based haptens to an exemplary alkylene oxide linker. Thehapten-linker conjugate then can be further derivatized as desired, ordirectly coupled to a carrier molecule, such as a protein carriermolecule.

With reference to Scheme 16, the exemplary benzodiazepine haptenincludes a hydroxyl group. This group was reacted with ethyliodoacetate,followed by treatment with sodium hydroxide to produce a compound havinga terminal carboxylic acid functional group. The carboxylic acid wasconverted to an NHS ester using EDAC as a coupling agent. The NHS esterwas reacted with either ethylene diamine or an exemplary bifunctionalPEG₈ linker. As a first option, the azoaryl compound can be reacted withethylene diamine to produce an amide having a terminal amine. Thiscompound can then be reacted with a maleimide-PEG-NHS ester to couple alinker to the hapten.

Alternatively, the reactive NHS ester can be reacted with a PEG₈ linkerto produce an amide having either a terminal carboxylic acid or hydroxylfunctional group. The carboxylic functional group of the amide can beconverted to an NHS ester using DCC as coupling agent. The NHS ester wasreacted with a BOC-protected hydrazine reagent, followed by deprotectionin 3M hydrochloric acid in dioxane, to produce the hydrazide.Alternatively, the NHS ester can be coupled to a protein carrier toproduce an immunogen.

The hydroxyl-terminated PEG conjugate can be reacted with mesylchloride, followed by reaction with a halide, such as iodide, to providethe halide-substituted compound. This compound can be reacted with theillustrated dimethyl amine carbodiimide to produce a compound useful fordirect labeling of a biomolecule.

13. Maleimide/Hydrazide PEG-Linker Synthesis

Scheme 17 shows a general method for preparing maleimide/hydrazideheterobifunctional PEG linkers. Briefly, a maleimide/active ester PEGlinker 102 (such as obtained from Quanta Biodesign) is reacted with aprotected hydrazine derivative 104 to produce compound 106. Compound 106was then deprotected with acid to yield the maleimide/hydrazide PEGlinker 108.

A specific synthesis of a maleimide/hydrazide PEG₄ linker is outlined inScheme 16 below. To the active ester 110 (116mg, 1.0 eq.) in 5 ml drydioxane was added 30 mg (1.0 eq.) of the Boc protected hydrazine 112 in5 ml of dry dioxane over 1 hour. The reaction was then stirred atambient temperature under dry nitrogen for 16 hours. The reactionmixture was fractionated by HPLC utilizing a Waters Delta 600 HPLCfitted with a 2996 photo-diode array detector and a Phenomenex luna 10μ, C18(2), 100A, 250×30 mm column. The column was eluted with 30-60% ACN/ water over 30 min at a flow rate of 12 mL / min. The desired Bocprotected-PEG₄-maleimide 114 eluted at 38 minutes giving 50 mg of athick yellow oil after drying under high vacuum. The final deprotectedhydrazide 116 was then obtained by stirring the residue with 6 ml ofanhydrous 2 N HCL/dioxane under dry nitrogen for 45 minutes.Concentration via rotary evaporation then gave 55 mg of thehydrazide-PEG₄-maleimide HCL salt.

14. Linker-Detectable Label Conjugates

Certain embodiments of the present invention concern forming conjugatesusing linkers. The following non-limiting examples are provided toillustrate embodiments of the method by reference to embodiments forforming detectable label conjugates using maleimide PEG active esters toexemplify the process. A person of ordinary skill in the art willappreciate that the illustrated embodiments can be used to form othertypes of conjugates as disclosed herein.

In one embodiment, a disclosed specific-binding moiety nanoparticleconjugate is prepared according to the processes described in Schemes 19to 22 below, wherein the heterobifunctional polyalkylene glycol linkeris a polyethylene glycol linker having an amine-reactive group (activeester) and a thiol-reactive group (maleimide). As shown in Scheme 19, ananoparticle (such as a quantum dot) that has one or more availableamine groups is reacted with an excess of the linker to form anactivated nanoparticle.

Thiol groups may be introduced to the antibody by treating the antibodywith a reducing agent such as DTT as shown in Scheme 20. For a mildreducing agent such as DTE or DTT, a concentration of between about 1 mMand about 40 mM, for example, a concentration of between about 5 mM andabout 30 mM and more typically between about 15 mM and about 25 mM, isutilized to introduce a limited number of thiols (such as between about2 and about 6) to the antibody while keeping the antibody intact (whichcan be determined by size-exclusion chromatography). A suitable amountof time for the reaction with a solution of a particular concentrationcan be readily determined by titrating the number of thiols produced ina given amount of time, but the reaction is typically allowed to proceedfrom 10 minutes to about one day, for example, for between about 15minutes and about 2 hours, for example between about 20 minutes andabout 60 minutes.

The components produced according to Schemes 19 and 20 are then combinedto give a conjugate as shown in Scheme 21.

Although Schemes 19-21 illustrate an optimal process for maleimide PEGactive esters, wherein the nanoparticle is first activated by reactingan amine group(s) with the active ester of the linker to form anactivated nanoparticle, it also is possible to first activate theantibody by reacting either an amine(s) or a thiol(s) on the antibodywith the linker and then react the activated antibody with thenanoparticle [having either a thiol(s) or an amine(s) to react with theremaining reactive group on the linker as appropriate].

Thus, in an alternative embodiment, an antibody is activated forconjugation and then conjugated to a nanoparticle as shown in Schemes 22and 23 below. In Scheme 23, the antibody is activated instead of thenanoparticle as was shown in Scheme 19. In the particular embodiment ofScheme 22, a sugar moiety (such as located in a glycosylated region ofthe Fc portion of the antibody) is first oxidized to provide an aldehydegroup, which is then reacted with an aldehyde-reactive group of thelinker (such as a hydrazide group of the illustrated maleimide/hydrazidePEG linker).

Then, as shown in Scheme 23, a thiol-reactive group of the linkerportion of the activated antibody (such as a maleimide group asillustrated) is then reacted with a thiol group on the nanoparticle.Again, the process can be reversed, wherein the linker is first reactedwith an aldehyde group on the nanoparticle (formed, for example, byoxidation of a sugar moiety) to form an activated nanoparticle, and thenthe activated nanoparticle can be reacted with a thiol group on theantibody.

Although schemes 17-2 above and 22 that follows show particular examplesof conjugates for illustrative purposes, it is to be understood that theratio of specific-binding moiety (in this case, antibody) tonanoparticle in the disclosed conjugates can vary from multiple (such as5, 10, 20 or more) specific binding moieties per nanoparticle tomultiple nanoparticles per specific-binding moiety (such as 5, 10, 20 ormore).

15. Introduction of Thiols to Antibodies

To activate an antibody for conjugation, for example, an anti-mouse IgGor anti-rabbit IgG antibody, the antibody can be incubated with 25 mmolDTT at ambient temperature (23-25° C.) for about 25 minutes. Afterpurification across a PD-10 SE column, DTT-free antibody, typically withtwo to six free thiols, is obtained (Scheme 2). The exemplary procedureoutlined for preparing goat anti-mouse IgG thiol is generally applicableto other antibodies. The number of thiols per antibody can be determinedby titration, for example, by using the thiol assay described in U.S.Provisional Patent Application No. 60/675,759, filed Apr. 28, 2005,which application is incorporated by reference herein.

16. Conjugates of Immunoglobulins and Streptavidin with CdSe/ZnS QuantumDots for Ultrasensitive (and Multiplexed) Immunohistochemical and InSitu Hybridization Detection in Tissue Samples.

One embodiment of a method for incorporating an immunoglobulin into aquantum dot shell is described in this example. This embodimentinvolves: 1) functionalization of amine-terminated quantum dot cappinggroups with a suitable heterobifunctional NHS ester-(PEG)x-maleimide;(x=4,8,12) 2) reduction of native disulfides by treatment withdithiothreitol (DTT); 3) derivatizing maleimide-terminated quantum dotswith these thiolated immunoglobulins; and 4) purifying the conjugatesusing suitable techniques, such as size-exclusion chromatography. Theprocess is depicted in Scheme 24.

A streptavidin conjugate can be made by substituting a thiolatedstreptavidin for the thiolated immunoglobulin in the process. Forexample, a streptavidin molecule treated with 2-iminothiolane.

The quantum dots used in this example were protected by anelectrostatically bound shell of trioctyl phosphine oxide (TOPO) and anintercalating amphiphilic polymer to induce water solubility. Thispolymer has approximately 30 terminal amine groups for furtherfunctionalization. See E. W. Williams, et. al. “Surface-ModifiedSemiconductive and Metallic Nanoparticles Having Enhanced Dispersibilityin Aqueous Media”, U.S. Pat. No. 6,649,138 (incorporated by reference,herein). In order to form highly sensitive quantum dot conjugates,antibodies were attached to the quantum dots with varying ratios. Thechemistry is similar to that described in U.S. Provisional PatentApplication No. 60/675,759, filed Apr. 28, 2005, which is incorporatedby reference herein.

This methodology is advantageous due to the need for few reagentsbecause native disulfides are used. Additionally, the antibody remainsdiscrete and does not form fragments. This allows for two binding sitesfrom each tethered antibody. Furthermore, highly stable and brightconjugates are produced. The brightness surpasses commercially availablestreptavidin-QD conjugates (Invitrogen Corporation, Eugene, Oreg.) onthe same tissue. Goat anti-biotin and rabbit anti-DNP antibodiesconjugated to quantum dots of differing wavelengths of emission wereproduced, thereby permitting multiplex assays. HPV detection throughFISH was demonstrated with the disclosed quantum dot conjugates.

VIII. Embodiments of a Method for Using Disclosed Haptens, HaptenConjugates, and Compositions Thereof

A. In Situ Hybridization

Certain exemplary embodiments of the present invention are disclosedherein with reference to the attached drawings. FIG. 1 illustrates oneembodiment of an in situ hybridization scheme 10 that can be implementedwith various embodiments of disclosed haptens. A sample having a target12, such as a protein, is selected. A probe 14 useful for detecting thetarget 12, such as an antibody, also is selected. At least one hapten 16of the classes of haptens disclosed herein is conjugated to the probe14. Target 12 is treated with the probe 14 conjugated to the hapten 16in a manner effective to form a complex that can be visualized using anysuitable means. FIG. 1 depicts treating the target 12 complexed with theprobe-hapten conjugate with an anti-hapten antibody 18 having adetectable label 20. A person of ordinary skill in the art willappreciate that the detectable label 20 can be any of the variety ofsignal generating moieties disclosed herein or that would be known to aperson of ordinary skill in the art, or combinations thereof, such as anenzyme, an organic chromophore, such as a fluorophore, chromophoricnanoparticles, such as fluorescent quantum dots, etc. The detectablelabel 20 is used to visualize the complex. For example, if thedetectable label 20 is an enzyme, a substrate for the enzyme isprovided, thereby producing a uniquely identifiable precipitate, such asa colored precipitate.

FIG. 1 also illustrates using at least one, and typically plural probes,where the probe or probes is conjugated to at least one hapten, andpotentially plural different haptens, to simultaneously visualizemultiple targets in a sample. FIG. 1 illustrates a sample having aparticular target 22 that is recognized by a probe 24. Hapten 26 isconjugated to the probe 24. Hapten 26 may be the same or different fromhapten 16. The sample is then treated with an anti-hapten antibody 28conjugated to a detectable label 30. This process can continue, asillustrated for targets 32 and 42.

Signal generating moieties 20, 30, 40 and 50 as illustrated in FIG. 1can be the same label, such as an enzyme. In this situation, the processmay comprise adding anti-hapten antibodies 18, 28, 38 and 48sequentially. After each application a different precipitate is formedby adding a different substrate. Substrates used for previousvisualization reactions are washed from the samples before second andsubsequent substrates are added.

FIG. 2 illustrates antibody 60 coupled to detectable label, such as anenzyme 62. An enzyme substrate 64 is added to produce a detectableenzymatic product 66. One specific embodiment of this process is SilverIn Situ Hybridization (SISH). One suitable enzyme 62 for SISH ishorseradish peroxidase, using silver ions and hydrogen peroxide as asubstrate. The detectable product 66 is elemental silver particles.

As another example, enzyme 62 might be alkaline phosphatase. Substrate64 is a source of silver ions and a phosphate-protected reductant.Again, the visually detectable product 66 is elemental silver. Silvercan be detected by any suitable means, including bright fieldmicroscopy.

The embodiment illustrated in FIG. 2 also can be used to implementChromogenic In Situ Hybridization. In this process, an enzyme 62 isagain selected, with suitable examples including those disclosed hereinor are otherwise known to those of ordinary skill in the art, withhorseradish peroxidase and alkaline phosphatase being used to exemplifyparticular embodiments. A substrate is then selected suitable forproducing a colored precipitate product 66 that can be detected usingtechniques known in the art, including bright field microscopy. Thechromogenic compound can be fluorogenic. Suitable fluorogenic compoundsare commercially available from various sources. For example,beta-lactamase and fluorogenic beta-lactamase substrates are availablefrom Invitrogen Detection. The substrate can be made fluorogenic by theenzymatic action, or a fluorogenic substrate can be renderednon-fluorescent. Quantum dots also can be used to visualizeimmunohistochemical interactions too. Fluorescent probes and quantumdots typically are monitored using a fluorescence microscope.

FIG. 3 illustrates one embodiment of a direct detection process. Forthis process, a primary antibody 70 is selected for a particular target.For example, the primary antibody 70 might be a monoclonal antibody,such as mouse monoclonal IgG antibody. Primary antibody 70 alsotypically includes a detectable label 72, as discussed above.

Alternatively, an amplification process can be used, as is illustratedschematically in FIG. 4. This embodiment also can be used for diagnostictests. A target is selected. A primary antibody 80 is added to thesample in a manner to allow complexation of the target and primaryantibody. A secondary antibody 82 against the primary antibody 80 isadded to the sample. Antibody 82 includes a detectable label that can beused to identify, particulary visually or by visual means, such asmicroscopy, the complexed target using a substrate, as discussed herein.Antibody 82 can be any suitable antibody, including by way of exampleand without limitation, a labeled rabbit anti-mouse IgG antibody.Moreover, a secondary antibody 86 to the primary antibody 80 also can beadded to the sample. Antibody 86 can be any suitable antibody againstthe primary antibody, such as an antibody from a different species. Forexample, antibody 86 might be, by way of example and without limitation,a goat antibody raised against the primary antibody, such as a mouse IgGantibodies. FIG. 4 illustrates adding at least one additionalanti-antibody 88 having a detectable label 90 to the sample to amplifythe signal produced by the detected target. In this exemplary process,the antibody 88 might be a labeled rabbit anti-goat IgG antibody.Antibody 88 can be added simultaneously with, or subsequent to, as thelabeled antibody 84.

Certain embodiments of the present invention are facilitated by usinganti-hapten monoclonal antibodies. FIG. 5 schematically represents oneembodiment of the present invention useful for hybridoma screening. Aswith preceding examples, a particular target is selected. For example, atarget situated in a tissue 100, such as the illustrated lambda epitope102, is identified. A primary antibody 104 directed to the target 102 isadministered in a manner effective for the antibody to recognize thetarget. One example of a suitable primary antibody 104 for theillustrated system is anti-Lambda rabbit antibody. As indicted in FIG.5, antibody 104 has at least one, and potentially plural, haptens 106conjugated thereto, as with the illustrated embodiment. A person ofordinary skill in the art will recognize first that the number ofhaptens conjugated to the antibody can vary, but this number typicallyis from 1 to about 5 haptens, but more typically is 2 to 3. Furthermore,a person of ordinary skill in the art will appreciate that the haptensconjugated to the primary antibody can be the same or different.

Tissue sample 100 is treated with anti-hapten antibodies 108. Forexample, in the embodiment illustrated in FIG. 5, haptens 106, coupledto the primary antibody 104, then effectively become coupled to ananti-hapten antibody 108, such as may be provided from a hybridoma mousemonoclonal antibody. Thus, for each hapten 106 coupled to the primaryantibody 104, there will be a secondary antibody 108. The complex formedby the anti-hapten antibody 108, such as a mouse monoclonal antibody,then must be identified. One method is to now treat the composition withan antibody that recognizes the mouse antibody, such as a goat antibody.In the illustrated embodiment of FIG. 5, goat antibodies 110 areconjugated to a detectable label, such as an enzyme, including theillustrated horseradish peroxidase (HRP) enzymes 112. This complex isthen incubated with an HRP substrate, as is known to persons of ordinaryskill in the art, to form detectable, e.g. colored, precipitates. Thisprocess can be used for screening, such as hybridoma screening.

To screen for antihapten monoclonal antibodies, a tissue sample, such asnormal human tonsil tissue is obtained. The sample may be embedded inparaffin, and if so, the tissue sample is deparaffinized, such as byusing VMSI EZPrep solution. Cell conditioning and antigen retrieval isthen performed using VMSI CC1. A primary polyclonal antibody, such ashuman anti-lamba (available from Dako), was conjugated to embodiments ofhaptens disclosed in the present application. Conjugation typicallyoccurred at the Fc region of the antibody. Conjugating to the Fc regionreduces the likelihood that the binding will affect the antibodyspecificity. A solution comprising an effective amount of the primaryantibody is applied to the tissue for an effective period of time. Forworking embodiments the effective concentration has been about 10 μg/mlof the primary antibody, and the effective time period has been about 60minutes. The tissue sample is then washed. Thereafter, a potentialanti-hapten antibody (e.g. KLH-CGT1-1.1+5-27F09-02E01) is applied to thetissue sample for an effective period of time, such as about 60 minutes.The antibody is then detected using any suitable means, such as VMSIOmni Map DAB stain.

Automated immunohistochemistry (IHC) screening of potential anti-haptenantibodies was performed using a VMSI Discovery XT and formalin-fixed,paraffin-embedded human tonsil tissue on glass slides. Tissue samplesfirst undergo de-pariffination, antigen retrieval, followed by theaddition of a primary antibody linked to the hapten of interest, thepotential anti-hapten antibody and a detection antibody. The detectionantibody is visualized using a chromogen detection reagent from VMSI.Stained slides were manually screened under a microscope. Samples havinga correct primary antibody staining pattern were selected as potentialanti-hapten candidates. To test for selectivity and specificity,candidate anti-hapten cell fusion products are further screened usingprimary antibodies conjugated to a hapten of a different chemical classunder the same staining method detailed above.

FIG. 6 is a photomicrograph depicting IHC positive staining ofanti-hapten antibody detection using a primary antibody conjugated to adisclosed embodiment of a nitropyrazole hapten according to the presentinvention. FIG. 6 clearly demonstrates visualization of a target in asample using haptens according to the present invention coupled to adetectable label.

FIG. 7 is a photomicrograph depicting IHC negative staining using ananti-hapten antibody, such as an anti-nitropyrazole antibody, and aprimary antibody conjugated to a disclosed embodiment of a class ofhaptens according to the present invention, such as a phenylthiourea.FIG. 7 clearly demonstrates visualization of a target in a sample usinghaptens according to the present invention coupled to a detectablelabel.

Embodiments of the present invention are useful for multiplexing, i.e.simultaneous detection of multiple targets in a sample. One embodimentof this approach is illustrated schematically in FIG. 8. FIG. 8illustrates that a sample, such as tissue sample 120, may have multipletargets, including: Ki-67 (122) [a protein antigen that accumulates fromG1-phase to mitosis, where it is found at its highest content. Duringinterphase the Ki-67 protein is predominantly associated with thenucleoli. During mitosis it shows a close association with thechromosomes. Ki-67 is present in nuclei of proliferating (G1-, S-,G2-phase and mitosis) cells, but not in nuclei of quiescent or restingcells (G0-phase). Recently it was demonstrated that the Ki-67 proteinbelongs to the family of MPM-2 antigens and that phosphorylation of theKi-67 protein during mitosis is associated with the condensation of thechromosomes and the separation of sister chromatids. A C-terminal domainof Ki-67 protein can bind to all three members of the mammalianheterochromatin protein 1 (HP1) family in vitro and in vivo suggesting arole for Ki-67 protein in the control of higher order chromatinstructure; CD3 antigen (124) [CD3 is a protein complex composed of threedistinct chains (CD3γ, CD3δ and CD3ε) in mammals, that associate withT-cell receptors (TCR) to generate an activation signal inT-lymphocytes. The TCR, ζ-chain and CD3 molecules together comprise theTCR complex. The CD3γ, CD3δ and CD3ε chains are highly related cellsurface proteins]; Kappa protein (126); CD20 (128) [an antigen expressedon normal and malignant human B cells that is thought to function as areceptor during B cell activation]; CD-68 antigen (130) [a 110 kDahighly glycosylated transmembrane protein which is mainly located inlysosomes]; and lambda protein (132). An antibody for each of thetargets 122, 124, 126, 128, 130 and 132 is then selected, and added tothe tissue sample 120 in a manner effective to allow antibodyrecognition of the target. For example, Ki-67 (122) may be recognized bya primary antibody 134 conjugated to BF hapten 136. An anti-BFmonoclonal antibody 138 is then added to the sample in a mannereffective to allow recognition of the BF hapten by the anti-BF antibody138. Anti-BF monoclonal antibody 138 includes a detectable label 140,such as Qdot 585. Color emission spectra for various Qdots are providedat www.niaid.nih.gov/vrc/prd/fig3_qdot_spectra.pdf. Qdot 585 produces ayellow-orange light. A person of ordinary skill in the art willappreciate that this process can be varied from that described. Forexample, hapten 136 need not be BF, nor does the detectable label 140need to be Qdot 585, nor even a Qdot. Rather, all various combinationsof haptens and signal generating moieties as described herein and aswould be known to a person of ordinary skill in the art can be used topractice the invention.

With continued reference to FIG. 7, CD3 (124) may be recognized by aprimary antibody 142 conjugated to biotin 144. This feature illustratesanother embodiment of the present invention where known agents, such asbiotin, can be used in combination with disclosed embodiments ofhaptens, hapten conjugates, and compositions thereof. An anti-biotinmonoclonal antibody 146 is then added to the sample in a mannereffective to allow recognition of biotin by the anti-biotin antibody146. Anti-biotin monoclonal antibody 146 includes a detectable label148, such as Qdot 525, which produces a bluish green color.

Kappa (126) may be recognized by a primary antibody 150 conjugated todinitrophenyl hapten 152. An anti-DNP monoclonal antibody 154 is thenadded to the sample in a manner effective to allow recognition of theDNP hapten by the anti-DNP antibody 154. Anti-biotin monoclonal antibody154 includes a detectable label 156, such as Qdot 605, which produces anorange color.

CD20 (128) may be recognized by a primary antibody 158 conjugated tonitrophenyl hapten 160. An anti-NP monoclonal secondary antibody 162 isthen added to the sample in a manner effective to allow recognition ofNP 160 by the anti-NP antibody 162. Anti-NP monoclonal antibody 162includes a detectable label 164, such as Qdot 655, which produces alight red color.

CD-68 (130) may be recognized by a primary antibody 166 conjugated to TShapten 168. An anti-TS monoclonal secondary antibody 170 is then addedto the sample in a manner effective to allow recognition of TS 168 bythe anti-TS antibody 170. Anti-TS monoclonal antibody 170 includes adetectable label 172, such as Qdot 565, which produces a light greencolor.

Lambda (132) may be recognized by a primary antibody 174 conjugated torotenone hapten 176. An anti-Rot monoclonal secondary antibody 178 isthen added to the sample in a manner effective to allow recognition ofRot 176 by the anti-Rot antibody 178. Anti-Rot monoclonal antibody 178includes a detectable label 180, such as Qdot 705, which produces a darkred color. Thus, by using different signal generating moieties, severaldifferent sample targets can be visualized substantially simultaneously,or sequentially, as may be desired.

Working embodiments have used multiple different haptens, and antibodiesthereto, to visualize a detectable target. FIG. 9 illustrates theresults of such an approach. FIG. 9 is a staining image produced usingmultiple haptens and antibodies thereto. FIG. 9 clearly showsvisualization of the protein.

Embodiments of the present invention also are useful for implementing adifferent type of multiplexing, i.e. simultaneous detection of multipledifferent types of targets, such as protein and nucleic acid targets, ina sample. This is illustrated schematically in FIG. 10 with reference toHer2 (human epidermal growth factor receptor 2). Her2 is a gene thathelps control how cells grow, divide, and repair themselves. The Her2proto-oncogene encodes a transmembrane glycoprotein of 185 kDa withintrinsic tyrosine kinase activity. Amplification of the Her2 gene andoverexpression of its product induce cell transformation. Numerousstudies have demonstrated the prognostic relevance of p185(Her2), whichis overexpressed in 10% to 40% of human breast tumors.

FIG. 10 illustrates fluorescent imaging. As illustrated in FIG. 10, ahapten labeled Her2 probe 200 is added to a sample in a manner effectiveto allow the probe to complex with the Her2 gene. Probe 200 includes ahapten 202 that can be any known hapten, including embodiments ofhaptens disclosed herein. FIG. 10 illustrates using dinitrophenyl hapten202. The complexed gene is then treated with an anti-hapten antibody204. Anti-hapten antibody 204 includes detectable label 206, such asQdot 565.

An anti-Her2 protein antibody 208, such as Anti-her2 4B5 rabbitantibody, is added to the sample in a manner effective to allowrecognition of the Her2 protein. The anti-Her2 antibody 208 includes atleast one hapten 210, and potentially plural haptens 210, which may bethe same or different. The embodiment illustrated in FIG. 10 illustratesthe process using biotin. An anti-hapten secondary antibody 212 is thenadded to the sample in a manner effective to allow complexation of thesecondary antibody 212 and hapten(s) 210. Anti-hapten secondary antibody212 includes a detectable label 214, such as a Qdot 655. Thus, theembodiment illustrated in FIG. 10 allows multiplexed detection of geneand gene product.

FIG. 11 illustrates the results of such a multiplexed chromogenicdetection. FIG. 11 is a staining image depicting detection of proteinand 2 genes, such as by using anti-biotin and anti-dinitrophenylantibodies.

IX. Test Kits

Disclosed embodiments of the present invention provide, in part, kitsfor carrying out various embodiments of the method of the invention.Examples of such kits include those useful for cholesterol analyses,pregnancy kits, cancer diagnostic kits, etc. Test kits of the presentinvention typically have a hapten conjugate according to the presentinvention, such as at least one hapten-specific binding moleculeconjugate, including hapten-antibody conjugates and/or hapten-nucleicacid probe conjugates, and an anti-hapten antibody, particularly ananti-hapten antibody conjugated to a detectable label

As a specific example, kits are provided for characterizing a mammaliantumor's responsiveness to drug therapies, such as inhibitors. Particularexamples include, without limitation, an inhibitor of the mTOR pathwayor a dual mTOR pathway inhibitor and an EGR pathway inhibitor comprisingat least two reagents, preferably antibodies, that can detect theexpression, phosphorylation, or both of polypeptides in the EGF pathway,the mTOR pathway, or both. For example, the kit can contain at leasttwo, three, or four reagents that bind to a phosphorylated form of ERK,that bind to the phosphorylated form of MEK, that bind to HIF-1α, orthat bind to mTOR. Further, the kit can include additional componentsother than the above-identified reagents, including but not limited toadditional antibodies. Such kits may be used, for example, by aclinician or physician as an aid to selecting an appropriate therapy fora particular patient.

X. Automated Embodiments

A person of ordinary skill in the art will appreciate that embodimentsof the method disclosed herein for using hapten conjugates can beautomated. Ventana Medical Systems, Inc. is the assignee of a number ofUnited States patents disclosing systems and methods for performingautomated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200,6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. publishedapplication Nos. 20030211630 and 20040052685, each of which isincorporated herein by reference.

Particular embodiments of hapten staining procedures were conductedusing various automated processes. Additional detail concerningexemplary working embodiments are provided in the working examples.

XI. Working Examples

The following examples are provided to illustrate certain specificfeatures of working embodiments. The scope of the present invention isnot limited to those features exemplified by the following examples.

Materials

DTT was purchased from Aldrich and quantum dots were purchased fromQuantum Dot, Co. and used as received. NH₂-dPEG₈-CO₂H, NH₂-dPEG₈-OH,NHS-dPEG₁₂-MAL and NHS-dPEG₄-MAL were purchased from Quanta BioDesign.Goat anti-biotin was received lyophilized from Sigma. Antibodyconcentrations were calculated using ε₂₈₀=1.4 ml mg⁻¹cm⁻¹. Quantum dotconcentrations were determined using ε_(601(±3))=650 000 M⁻¹cm⁻¹ for 605nm emitting quantum dots (QD₆₀₅) and ε_(645(±3))=700 000 M⁻¹cm⁻¹ for QD655. Deionized water was passed through a Milli-Q Biocel System to reacha resistance of 18.2 MΩ. Buffer exchange was performed on PD-10 columns(GE Biosciences). Size-exclusion chromatography (SEC) was performed onAkta purifiers (GE Biosciences) which was calibrated with proteinstandards of known molecular weight. The flow rate was 0.9 ml/min on aSuperdex 200 GL 10/300 (GE Biosciences) running PBS, pH 7.5.

Example 1

This example concerns the synthesis of Rotenone isoxazoline.

Rotenone (20.00 g, 50.7 mmol, 1.0 eq.) and hydroxylamine hydrochloride(35.20 g, 507 mmol, 10.0 eq.) were suspended/dissolved in absoluteethanol (600 mL). A solution of sodium hydroxide (24.30 g, 608 mmol, 12eq.) in water (120 mL) was added to the stirred suspension and refluxedfor three hours. After the reaction was cooled to room temperature, thesolution was filtered and the filtrate reduced in vacuo to approximately150 mL volume. The reduced filtrate was diluted with water (200 mL) andextracted three times with methylene chloride (200 mL each). Themethylene chloride washes were combined, dried over anhydrous magnesiumsulfate, filtered and the solvent removed under vacuum. The resultingmaterial (20 g) was taken up in methylene chloride (10 mL) and purifiedby flash chromatography (Isco Combiflash) using a 330 gram column andeluting using a methylene chloride to 20% methanol/methylene chloridegradient. The desired compound (6.17 g, 30%) was isolated as the earliereluting fraction. Purity was determined by HPLC and structure by¹H/¹³C-NMR and MS.

Example 2

This example concerns the synthesis of Rotenone isoxazoline acetic acid.

Rotenone isoxazoline (3.53 g, 8.62 mmol, 1.0 eq.) was stirred/suspendedin anhydrous dimethylformamide (80 mL). Bromoacetic acid (28.10 g, 86.2mmol, 10.0 eq.) was added under nitrogen. Cesium carbonate (28.1 g, 86.2mmol, 10.0 eq.) and silver oxide (2.99 g, 12.9 mmol, 1.5 eq.) were addedand the reaction stirred under nitrogen at ambient temperature for 21hours. The reaction mixture was diluted with methylene chloride (120mL), filtered and the solvent removed in vacuo. The residue was taken upin methylene chloride (15 mL) and chromatographed (Isco CombiFlash)using a 120 gram Redisep column with a 0 to 10% methanol gradient inmethylene chloride. The fractions containing the desired compound werecombined and concentrated under vacuum to give 2.98 g (74%). Purity wasdetermined by HPLC and structure by ¹H/¹³C-NMR and MS.

Example 3

This example concerns the synthesis of diazapinone ester.

1,3-Dihydro-4-(2-hydroxyphenyl)-2H-1,5-benzodiazapin-2-one (2.837 g,11.2 mmol, 1.0 eq) Was stirred-suspended in 40 ml of DMF, 34 ml (34mmol, 3.0 eq) of a 1.0 M (in THF) solution of Potassium tert-butoxideadded, 1.6 ml (13.5 mmol, 1.2 eq) of ethyl iodoacetate added, andreaction Stirred under N2 for 3 hours. Then an additional 1.6 ml (13.5mmol, 1.2 eq) of ethyl iodoacetate was Added, and stirring continued for2 hours. The reaction was then poured into 100 ml of water and Extractedwith etoac (3×100 ml). The etoac extracts were combined, dried overmgso4, solvent Removed in vacuo, and resulting oil purified by flashchromatography, eluting with etoac/Hexane (20/80). Obtained 806 mg (21%yield). Purity was determined by HPLC and structure by NMR and MS.

Example 4

This example concerns the synthesis of diazapinone acid.

The diazapine ester (999 mg, 2.95 mmol, 1.0 eq) was dissolved in 30 mLof MeOH, 944 mg (23.6 mmol, 8.0 eq) of sodium hydroxide dissolved in 15mL of water added, and reaction stirred for 40 minutes. The reaction wasthen diluted with 75 mL of water, pH adjusted to less than 4 with 6 MHCl, and extracted with EtOAc (3×75 mL). The EtOAc extracts werecombined, dried over MgSO4, and solvent removed in vacuo. Obtained 865mg (94% yield). Purity was determined by HPLC and structure by NMR andMS.

Example 5

This example concerns the synthesis of oxopodophyllotoxin frompodophyllotoxin.

Podophyllotoxin (3.15 g, 7.61 mmol, 1.0 eq) was dissolved in 50 mL ofDCE, manganese(IV) oxide (6.6 g, 76 mmol, 10 eq.) added, and thereaction mixture was refluxed for 1 hour. Additional manganese(IV) oxide(6.6 g, 76 mmol, 10 eq.) was added, and refluxing continued for 5 morehours, then reaction stirred at room temperature for 60 hours. Thereaction was then filtered through celite to give a red-brown solution,filtrate solvent removed in vacuo, and resulting residue recrystallizedfrom EtOH. Obtained 1.968 g (63% yield). Purity was determined by HPLCand structure by NMR and MS.

Example 6

This example concerns the synthesis of pyrazopodophyllic acid.

Oxopodophyllotoxin (200 mg, 0.485 mmol, 1.0 eq) was stirred-suspended in10 mL of EtOH. Methoxyphenyl hydrazine hydrochloride (110 mg, 0.631mmol, 1.3 eq) was added, pyridine (0.300 mL, 3.71 mmol, 7.6 eq) wasadded, and the reaction mixture stirred under N₂, at 95° C., for 18hours. The reaction was then allowed to cool, poured into 20 mL ofsaturated sodium bicarbonate, and extracted with EtOAc (3×20 mL). TheEtOAc extracts were combined, dried over MgSO₄, solvent removed invacuo, and resulting residue purified by flash chromatography, elutingwith DCM/MeOH (98/2). Obtained 121 mg (47% yield). Purity was determinedby HPLC and structure by NMR and MS.

Example 7

This example concerns an exemplary synthesis of hapten carboxylic acidN-hydroxysuccinimidyl esters.

The hapten carboxylic acid (5.0 mmol, 1.0 eq.) was taken up in 10 ml ofdry DCM in a 50 ml round bottom flask. The solution was blanketed withdry nitrogen and NHS (5.5 mmol, 1.1 eq.) was added followed by 1.0 M DCCin DCM (6.0 mmol, 1.2 eq.), and triethylamine (6.0 mmol, 1.2 eq.). Thereaction was allowed to stir at room temperature under dry nitrogen for16 hours at which point the solvent was removed under vacuum. Theresidue was taken up in 2 ml of dry DCM and filtered to remove the ureabyproduct. The filter cake was then washed 2 times with 0.5 ml of dryDCM. The combined DCM portions were then dried under vacuum to give thehapten NHS ester which was used without further purification.

Example 8

This example concerns an exemplary synthesis of hapten-dPEG₈-carboxylicacids.

The hapten NHS ester or hapten acyl chloride (5.0 mmol, 1.0 eq.) wastaken up in 10 ml of dry DCM in a 50 ml round bottom flask. The solutionwas blanketed with dry nitrogen and amino-dPEG₈-carboxylic acid (5.5mmol, 1.1 eq.) was added followed by triethylamine (6.0 mmol, 1.2 eq.).The reaction was allowed to stir at room temperature under dry nitrogenfor 16 hours at which point the solvent was removed under vacuum. Theresidue was taken up in minimal methanol and purified by preparativeHPLC. The appropriate fractions were then pooled and dried under highvacuum to give the pure hapten-dPEG₈-acid.

Alternatively, haptens that have a sulfonyl chloride moiety, such as thethiazole-based haptens in Scheme 10 and the azoaryl-based haptens inScheme 15, could be directly coupled to amino-dPEG₈-carboxylic acidunder the same stoichiometry and reaction conditions to produce haptensulfamide-dPEG₈-carboxylic acid.

Example 9

This example concerns an exemplary synthesis of hapten-dPEG₈-carboxylicacid N-hydroxysuccinimidyl esters.

The hapten-dPEG₈-carboxylic acid (5.0 mmol, 1.0 eq.) was taken up in 10ml of dry DCM in a 50 ml round bottom flask. The solution was blanketedwith dry nitrogen and NHS (5.5 mmol, 1.1 eq.) was added, followed by 1.0M DCC in DCM (6.0 mmol, 1.2 eq.), and triethylamine (6.0 mmol, 1.2 eq.).The reaction was allowed to stir at room temperature under dry nitrogenfor 16 hours at which point the solvent was removed under vacuum. Theresidue was taken up in 2 ml of dry DCM and filtered to remove the ureabyproduct. The filter cake was then washed 2 times with 0.5 ml of dryDCM. The combined DCM portions were then dried under vacuum to give thehapten-dPEG₈-NHS ester which was used without further purification.

Example 10

This example concerns an exemplary synthesis of hapten-dPEG₈-hydrazides.

The hapten-dPEG₈-NHS ester (1.0 mmol, 1.0 eq.) was taken up in 5 ml ofdry DCM in a 25 ml round bottom flask. The solution was blanketed withdry nitrogen and BOC hydrazide (1.2 mmol, 1.2 eq.) was added. Thereaction was allowed to stir at room temperature under dry nitrogen for16 hours at which point the solvent was removed under vacuum. Theresidue was then taken up in 10 ml of 4N HCl in dioxane and stirred atroom temperature for three hours. The solvent was then removed undervacuum and the residue purified by preparative HPLC to give the purehapten-dPEG₈-hydrazide.

Example 11

The following examples concern an exemplary synthesis ofhapten-ethylamines by reacting ethylene diamine with a hapten-NHS,-sulfonyl chloride, -acid chloride or 1-fluoro-2,4-dinitrobenzene.

The hapten-NHS ester, hapten-sulfonyl chloride, hapten-acid chloride, or1-fluoro-2,4-dinitrobenzene (1 mmol, 1.0 eq.) was dissolved in anhydrousmethylene chloride (10 mL) and added dropwise to a solution of ethylenediamine (20 mmol, 20 eq.) in anhydrous methylene chloride (10 mL) undernitrogen and ambient conditions. The reaction mixture was stirred forone hour and the solvent removed in vacuo. The residue was taken up inan appropriate solvent and chromatographed on flash silica gel or bypreparative HPLC. Typical yields were 30-60%. Purity was determined byHPLC and structure by ¹H/¹³C-NMR and MS.

Example 12

This examples concerns reacting N-butoxycarbonyl ethylene diamine with ahapten-NHS ester, -sulfonyl chloride, -acid chloride or1-fluoro-2,4-dinitrobenzene.

The hapten-NHS ester, hapten-sulfonyl chloride, hapten-acid chloride, or1-fluoro-2,4-dinitrobenzene (1.0 mmol, 1.0 eq.) was dissolved inanhydrous methylene chloride (10 mL) and added dropwise to a solution ofN-butoxycarbonyl ethylene diamine (1.0 mmol, 1.0 eq.) in anhydrousmethylene chloride (10 mL) under nitrogen and ambient conditions. Thereaction mixture was stirred for two hours and the solvent removed invacuo. The residue was taken up in an appropriate solvent andchromatographed on flash silica gel or by preparative HPLC. Typicalyields were 20-40%. Purity was determined by HPLC and structure by¹H/¹³C-NMR and MS.

Example 13

This example concerns deprotecting hapten-BOC-ethylene diaminecompounds.

The Hapten-BOC-ethylene diamine (1.0 mmol, 1.0 eq.) was dissolved inanhydrous methylene chloride (2.0 mL). Trifluoroacetic acid (2.0 mL) wasadded under ambient conditions and stirred for 30 minutes. The solventwas removed under vacuum to constant weight and the material usedwithout purification. Typical yields were 90-95%. Purity was determinedby HPLC and structure by ¹H/¹³C-NMR and MS.

Example 14

This example concerns an exemplary synthesis of hapten-dPEG₈-maleimides.

The hapten-ethylene diamine derivative (1.0 mmol, 1.0 eq.) was dissolvedin anhydrous dimethyl formamide (5.0 mL) and triethylamine (4.0 mmol,4.0 eq.) was added and stirred at ambient conditions under nitrogen.MAL-dPEG₈-NHS (1.0 mmol, 1.0 eq., Quanta BioDesign) was dissolved inanhydrous dimethyl formamide (5.0 mL) and added to the hapten-ethylenediamine solution. The reaction was stirred at ambient conditions undernitrogen overnight. The solvent was removed under vacuum and purified bypreparative HPLC. Typical yields were 70-90%. Purity was determined byHPLC and structure by ¹H/¹³C-NMR and MS.

Example 15

This example concerns an exemplary synthesis of hapten-dPEG₇-alcohols.

In a 25 ml RB flask, the hapten-NHS ester, hapten-sulfonyl chloride,hapten-acid chloride, or 1-fluoro-2,4-dinitrobenzene (1.0 eq., 2.7 mmol)is reacted with amino-dPEG₇-alcohol (1.0 eq, 2.7 mmol, Quanta BioDesign)in 5 ml of dry DMF. The reaction is then blanketed with dry nitrogen andstirred at room for 16 hours. The solvent is removed under vacuum andthe target alcohol purified by either silica gel chromatography orpreparative HPLC. Product purity and identity was determined by HPLC,MS, and ¹H/¹³C-NMR.

Example 16

This example concerns an exemplary procedure of hapten-dPEG₇-mesylates.

The hapten-dPEG₇-alcohol was taken up in anhydrous DMF (7 mL) in a 25 mlRB flask. The flask was purged with dry nitrogen and mesyl chloride (1.1eq.) was added via syringe. The solution was stirred at room temperaturefor two minutes before adding anhydrous triethylamine (2.2 eq.) overapproximately 20 minutes. The reaction was stirred for 16 hours at roomtemperature before removing the solvent under vaccum. The residue wastaken up in dry DCM and purified via silica gel chromatography to affordthe mesylate after removing the solvent under vacuum. Product purity andidentity was determined by HPLC, MS, and ¹H/¹³C-NMR.

Example 17

This example concerns an exemplary procedure of hapten-dPEG₇-iodides.

The hapten-dPEG₇-mesylate was dissolved in dry acetone (10 mL) andconverted to the iodide by refluxing in the presence of sodium iodide(10 eq.) for three hours. The pure iodide was obtained after silica gelchromatography. Product purity and identity was determined by HPLC, MS,and ¹H/¹³C-NMR.

Example 18

This example concerns an exemplary procedure ofhapten-dPEG₇-carbodiimides.

The hapten-dPEG₇-alcohol was taken up in anhydrous DMF (7mL) in a 25 mlRB flask. The flask was purged with dry nitrogen and mesyl chloride (1.1eq.) was added via syringe. The solution was stirred at room temperaturefor two minutes before adding anhydrous triethylamine (2.2 eq.) overapproximately 20 minutes. The reaction was stirred for 16 hours at roomtemperature before removing the solvent under vacuum. The residue wastaken up in dry DCM and purified via silica gel chromatography to affordthe mesylate after removing the solvent under vacuum. Product purity andidentity was determined by HPLC, MS, and ¹H/¹³C-NMR.

Example 19

This example concerns an exemplary procedure of generating a4-amino-deoxycytidine triphosphate-dPEG₈-hapten.

4-Amino-deoxycytidine triphosphate (1.0 eq. as the triethylammoniumsalt) was dissolved in anhydrous DMSO to produce a 0.01M solution. Asolution of the hapten-dPEG₈-NHS (1.1 eq) in anhydrous DMSO was added tothe 4-amino-deoxycytidine triphosphate and stirred for 16-24 hrs. undernitrogen. The 4-amino-deoxycytidine triphosphate-dPEG₈-hapten waspurified by preparative HPLC using a Waters Sunfire OBD Preparativecolumn (10 μm, C18, 50×250 mm) and eluting with a gradient ofacetonitrile:water:0.8M triethylammonium carbonate (1:83:16 to 25:59:16over 30 min). The pure fractions were combined, lyophilized, andredissolved in a minimal amount of DI water. The water solution waspassed through a sodium ion-exchange column (SP Sephadex C-25, GELifesciences). The sodium salt of 4-amino-deoxycytidinetriphosphate-dPEG₈-hapten was lyophilized to constant weight, andcharacterized by HPLC, ¹H/¹³C-NMR and MS.

Example 20

This example concerns an exemplary procedure of generating an immunogenwith an immunogenic carrier protein and hapten-dPEG₈-NHS. A lyophilizedimmunogenic carrier protein, such as keyhole limpet hemocyanin (KLH),bovine thyroglobulin (BtG), or bovine serum albumin (BSA), wasreconstituted in 1.0 mL PBS, pH 7.2 to give approximately a 10 mg/mLprotein solution. The hapten-dPEG₈-NHS (300 eq. for KLH, 150 eq. forBtG, or 60 eq. for BSA) was dissolved in 100 μL DMF, added to theprotein solution and rotated at room temperature overnight. The reactionwas passed through a 0.2 μm GHP syringe filter and purified by SECchromatography on an AKTA Purifier running at 0.9 mL/min. over a GELifesciences Superdex 200 10/300 GL column with PBS, pH 7.2. Fractionswere pooled and collected corresponding to the monomeric immunogenicprotein. The hapten-labeled protein was characterized by BCA proteinassay (Pierce) for protein concentration and fluorescamine lysine assay(Bio-Tek) for hapten loading.

Example 21

This example concerns an exemplary procedure of conjugating a primaryantibody with a hapten-dPEG₈-NHS. A polyclonal or monoclonal antibody inPBS, pH 7.0-7.5, was treated with a solution of hapten-dPEG₈-NHS (20eq.) in anhydrous DMSO to give a final DMSO concentration not to exceed10% v/v. The reaction was rotated 18 hours in an amber vial at roomtemperature and filtered (0.2 μm GHP syringe filter) prior topurification by SEC chromatography on an AKTA Purifier running at 0.9mL/min. over a GE Lifesciences Superdex 200 10/300 GL column with PBS,pH 7.2. Typical yields were 70-80% with hapten coverage of 4-6 haptensper antibody.

Example 22

This example concerns an exemplary procedure of conjugating to theFc-region on a primary antibody with a hapten-dPEG₈-NHS. A polyclonal ormonoclonal antibody in PBS, pH 7.0-7.5, was treated with an unbuffered,aqueous solution of 100 mM sodium periodate to give a finalconcentration of 20 mM periodate. The solution was rotated at roomtemperature in an amber vial for two hours. The antibody was desaltedand buffer exchanged by passing through a Sephadex G-25 column (PD-10,GE Lifesciences) with ABS (0.1M acetate, 0.15M NaCl, pH 5.5). Theoxidized antibody solution was reacted with an unbuffered, aqueoussolution of polyacrylamide hydrazide (50 eq) (further detail concerningusing polyacrylamide hydrazide is provided by assignee's copendingapplication No. 60/931,546, which was filed on May 23, 2007, and isincorporated herein by reference) and incubated for one hour at ambienttemperature. Sodium cyanoborohydride (100 eq.) was added and thereaction was rotated overnight. The PAH-Ab conjugate was purified by SECchromatography on an AKTA Purifier running at 0.9 mL/min. over a GELifesciences Superdex 200 10/300 GL column with ABS, pH 5.5. Thehapten-dPEG₈-NHS (20 eq.) in anhydrous DMSO was added to give a finalDMSO concentration not to exceed 10% v/v. The hapten-dPEG₈-PAH-Abconjugate was purified by SEC chromatography on an AKTA Purifier runningat 0.9 mL/min. over a GE Lifesciences Superdex 200 10/300 GL column withPBS, pH 7.2.

Example 23

This example concerns an exemplary procedure of conjugating to theFc-region on a primary antibody with a hapten-dPEG₈-hydrazide. Apolyclonal or monoclonal antibody in PBS, pH 7.0-7.5, was treated withan unbuffered, aqueous solution of 100 mM sodium periodate to give afinal concentration of 20 mM periodate. The solution was rotated at roomtemperature in an amber vial for two hours. The antibody was desaltedand buffer exchanged by passing through a Sephadex G-25 column (PD-10,GE Lifesciences) with ABS (0.1 M acetate, 0.15 M NaCl, pH 5.5). Theoxidized antibody solution was reacted for one hour at room temperaturewith a solution of the hapten-dPEG-hydrazide (20 eq.) in DMSO, such thatthe final concentration of DMSO did not exceed 10% v/v. Sodiumcyanoborohydride (100 eq.) was added and the reaction was rotatedovernight. The hapten-dPEG₈-Ab conjugate was purified by SECchromatography on an AKTA Purifier running at 0.9 mL/min. over a GELifesciences Superdex 200 10/300 GL column with PBS, pH 7.2.

Example 24

This example concerns an exemplary procedure of conjugating a primaryantibody with a hapten-dPEG₈-maleimide. To a solution of polyclonal ormonoclonal antibody in 100 mM phosphate, 1 mM EDTA, pH 6.5 buffer wasadded DTT at a final concentration of 25 mM. This mixture was rotatedfor precisely 25 minutes before desalting on a Sephadex G25 (PD-10, GELifesciences) in 100 mM phosphate, 1 mM EDTA, pH 6.5 buffer.Hapten-dPEG₈-maleimide (50 eq.) was added as a DMF solution, such thatthe final concentration of DMF did not exceed 10% v/v. The reactionmixture was rotated overnight in an amber vial under ambient conditions.The hapten-dPEG₈-Ab conjugate was purified by SEC chromatography on anAKTA Purifier running at 0.9 mL/min. over a GE Lifesciences Superdex 20010/300 GL column with PBS, pH 7.5.

Example 25

This example concerns an exemplary procedure of conjugatingsingle-stranded DNA with a hapten-dPEG₈-carbodiimide. DNA (100 μg) wastaken up in TE buffer at 1 mg/ml in a tube and heated to 98° C. for oneminute. The reaction mixture was frozen in dry ice-ethanol and 100 μL0.5 M borate buffer, pH 9.5 was added. The reaction mixture was warmedto room temp and the hapten-dPEG₈-EDC (100 μL of 1.0 mM in DMSO) wasadded. The mixture was incubated at 60° C. for one hour, then added saltand precipitated with isopropanol. The precipitate was washed threetimes with 80% EtOH.

The results of the ssDNA labeling with DNP-dPEG₈-EDC are provided inFIGS. 17 and 18. FIG. 17 indicates that the percent of nucleotidelabeling increases, substantially linearly, with increased haptenconjugate. FIG. 17 illustrates that the percent nucleotide labeleddecreased with increasing DNA stock concentration.

Example 26

This example concerns an exemplary procedure of labeling DNA with ahapten-dPEG₈-amino-dCTP. The incorporation of the hapten label onto DNAwas accomplished by the nick translation procedure described in Rigby, PW; Dieckmann, M; Rhodes, C. and Berg, P., “Labeling deoxyribonucleicacid to high specific activity in vitro by nick translation with DNApolymerase I”, J. Mol. Biol., V113:237-251, 1977. Labeling efficiencywas 2-6% based on comparison of the 260 nm absorbance of DNA and λ_(max)and ε (extinction coefficient) of the specific hapten.

Example 27

This example concerns an exemplary procedure of screening anti-haptenhybridomas. Benzofurazan-dPEG₈-BSA (VMSI-1357-98) was coated ontomicroplates. The dilution buffer used was 0.15 molar phosphate bufferedsaline (PBS). The concentration of the Benzofurazan-dPEG₈-BSA was 2μg/ml, and the well concentration was 50 μL. These samples wereincubated at 4° C. overnight. 1% Nonfat dry milk (NFDM) (10 mg/mL, 300μL/well) was used as a blocking reagent, followed by incubation at 37°C. for 120 minutes. Plates were washed, as deemed necessary, using 0.15M PBS comprising 0.05% Tween 20. Each tested hapten then was used toproduce mouse antisera. A total concentration of 80 μL per well mouseantisera diluted with 1% NFDM in 0.15 M PBS was provided using thedilutions and plate design protocol indicated below in Table 6. Theplates were then incubated at 37° C. for 150 minutes. A goatantimouse-horseradish peroxidase conjugate (Gt-α-Mu-HRP, Pierce) wasused as a secondary antibody at a concentration of 1:10,000 in 0.15 MPBS with 0.05% Tween 20 to provide a total well volume of 50 μL/well.The plates were then incubated for 60 minutes at 37° C. The ELISA set upand results are summarized below in Table 2.

TABLE 2 ELISA Results Accession: 500421 Customer: Ventana Sample tested:mouse antisera Assay parameters: Step Reagent Serial dilution Dilutionbuffer Concentration Volume/well Incubation Ag coating VMSI-1357-98 —0.15M PBS  2 μg/mL 50 μL ON @ 4 C. Blocking 1% NFDM — 0.15M PBS 10 mg/mL300 μL  2 hr. @ 37 C. Sample Dilution see below 5 X 1% NFDM in 0.15M PBSstarting @ 1:50 80 μL 2.5 hr. @ 37 C. Secondary Ab Gt-α-Mu-HRP — .15MPBS w/0.05% Tween20 1:10000 50 μL 1 hr. @ 37 C. Plate design: Sampledilution 1:50 1:250 1:1250 1:6250 1:31250 1:156250 1 2 3 4 5 6 7 8 9 1011 12 Mu #1 A 1.89 0.01 1.81 0.00 1.82 0.00 1.60 0.00 1.06 0.00 0.560.00 Mu #2 B 1.76 0.01 1.87 0.00 1.73 0.00 1.25 0.00 0.60 0.00 0.25 0.00Mu #3 C 1.70 0.01 1.83 0.00 1.87 0.00 1.50 0.00 0.95 0.01 0.37 0.00 Mu#4 D 1.75 0.01 1.73 0.02 1.83 0.00 1.49 0.01 0.98 0.00 0.42 0.00 Mu #5 E1.62 0.01 1.58 0.00 1.72 0.00 1.35 0.00 0.74 0.00 0.33 0.00 Prebleedpool F 0.06 0.01 0.01 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 x x xx x x Antigen: VMSI-1357-98 Lot #C05081610 Wash Buffer: 0.15M PBS with0.05% Tween 20 Secondary Ab: HRP-goat-α-mu IgG Fc specific min. x-react#60312 Substrate: TMB lot #P502807 x = blank (no antigen) NFDM = non-fatdried milk Bleed date: Sep. 12, 2005 Assay date: Sep. 13, 2005The results shown in Table 2 indicate that each of the mice tested issuitable for raising an antibody response, and further that such haptenscan be visualized to confirm a response. With respect to the particularhapten tested, mouse number 1 appears to provide the best response overall dilutions tested.

Example 28

This example concerns an exemplary procedure of conjugation ofanti-hapten antibodies to horseradish peroxidase (HRP). Images producedusing such conjugates are provided by FIGS. 19-24.

Activation of HRP

To a 4 mL amber vial was added 78.8 mg (100 eq.) of MAL-dPEG₄™ NHS ester(Quanta Biodesign, Powell, Ohio, F.W.=513.50), followed by 2.46 mL (61.5mg, 1.53 μM) of HRP (Horseradish Peroxidase, Pierce, Rockford, Ill. LotFJ925901) as a 25 mg/mL solution in 0.1 M sodium phosphate, pH 7.5. Thevial was then placed on an autorotator in the dark at ambienttemperature (23-25° C.), and the amide bond forming reaction was allowedto proceed for one hour. A 400 μl aliquot was then removed forpurification, and the remainder of the solution was temporarily storedat 4° C. Pure HRP-PEG₄-maleimide was then obtained by fractionating thesample on an AKTA Purifier fitted with a Superdex 10/300 column (GELifesciences) eluted with 0.1 M sodium phosphate, pH 7.5 at 1.0 mL/min.The HRP containing fractions were pooled to give 2.0 ml of a 4.52 mg/mLsolution of HRP-PEG₄-maleimide (90% recovery) as measured by UV/VISspectrophotometry using an extinction coefficient at 280 nm of a 1%solution (pH 6.5) of 6.52.

Introduction of Thiols to Antibodies

To an 8 mL amber vial was added 3.0 mL of a mouse anti-hapten monoclonalantibody as a 2.1 mg/mL solution in 0.1 M sodium phosphate, 1.0 mM EDTA,pH 6.5. To this solution was then added 216 μL of a freshly prepared 500mM solution of the reducing agent DTT (1,4-Dithiothreitol,Sigma-Aldrich, St. Louis, Mo.). The vial was placed in the dark on anautorotator and the disulfide reduction was allowed to proceed for 25minutes. The reaction solution was split into four equal volumes (due tothe limited capacity of a desalting column used), and excess DTT wasremoved by passing each of the fractions across a PD-10 desalting column(GE Lifesciences) eluted with 0.1 M sodium phosphate, 1.0 mM EDTA, pH6.5. The antibody containing fractions were combined to give 8.0 mL of a0.8 mg/mL solution of reduced mouse anti-hapten antibody (71% recovery)as measured by UV/VIS spectrophotometry using an extinction coefficientat 280 nm of a 1% solution at pH 6.5 of 14.

HRP-Antibody Conjugation

To the reduced antibody (such as mouse anti-nitropyrazole monoclonalantibody), is added a three fold molar excess of HRP-PEG₄-maleimide. Thereaction is then incubated at ambient temperature (23-25° C.) for 16hours. After purification across a Superdex 200 10/300 GL SE column aconjugate, typically with an average of 2 or 3 HRPs per antibody, isobtained. The number of HRPs per antibody was determined by measuringthe ratio of absorbances at 280 nm/403 nm of the conjugate. Theconjugate was then stored in a cold room at 4° C. until use.

Example 29

This example concerns an exemplary procedure of conjugation ofanti-hapten antibodies to quantum dots (QD).

Reduction of Inter-Chain Disulfides on Antibodies

To a solution of polyclonal or monoclonal antibody in 100 mM phosphate,1 mM EDTA, pH 6.5 buffer was added DTT at a final concentration of 25mM. This mixture was rotated for precisely 25 minutes before desaltingon a Sephadex G25 (PD-10, GE Lifesciences) in 100 mM phosphate, 1 mMEDTA, pH 6.5 buffer.

Synthesis of QD-dPEG₁₂-MAL

To a solution of quantum dots (8-9 μM in 50 mM borate buffer, pH 8.0)was added NHS-dPEG₁₂-MAL (50 eq.) and rotated for two hours. Themaleimide-functionalized quantum dots (QD-dPEG₁₂-MAL) were purified bydesalting on a Sephadex G25 column (PD-10, GE Lifesciences) in 0.1 Mphosphate, 0.1 M NaCl, pH 7.0 buffer.

Synthesis of QD-MAL-Antibody Conjugate

The purified QD-maleimide was combined with the purified thiolatedantibody in molar ratios of 4:1 antibodies to QD and rotated for a 16hour period. The QD-Ab conjugate was purified by SEC chromatography onan AKTA Purifier running at 0.9 mL/min. over a GE Lifesciences Superdex200 10/300 GL column with 50 mM borate buffer, pH 8.0.

Example 30

This example demonstrates the ability of primary antibody-haptenconjugates to be visualized by chromogenic immunohistochemistry (IHC).Stainings representative of this approach are provided by FIGS. 19-24.FIG. 6 also is a staining representative of this approach, where methodsfor conjugating a primary antibody with a hapten are described inExamples 21-24. FIG. 7 is a control, whereby tissue treated with anantibody other than the appropriate anti-hapten antibody. FIG. 7establishes the specificity of the method as no visualization occursunless the appropriate anti-hapten antibody is used.

Tonsil tissue sections were treated with an anti-lambda polyclonalantibody (Dako) conjugated with haptens by the method in Examples 21,22, 23 or 24. The slides were developed using standard protocols for HRPsignal generation (by addition of DAB) on an automated stainer(BenchMark® XT, Ventana Medical Systems, Inc., Tucson, Ariz.). A typicalautomated protocol includes deparaffinization, several rinse steps,addition of a reaction buffer, addition of the primary antibody(anti-lambda:hapten conjugate), addition of the secondary antibody(anti-hapten:HRP conjugate), addition of DAB and hydrogen peroxide, andaddition of a counterstain.

Manual scoring was conducted by Board-certified pathologists. Stainingintensities, percentage of reactive cells, and cellular localizationwere recorded. For qualitative stain intensity, 0 is the most negativeand 3+ is the most positive. Slides were reviewed and scored by thepathologist prior to quantitation by optical imaging.

For optical imaging, a digital application (VMSI) with imagequantification based on the intensity (expressed as average opticaldensity, or avg. OD) of the stain converted to a numerical score wasutilized. A high-resolution image was captured for each sample and theOD value was determined based on specific classifiers for the shape andcolor range for positively stained cells. At least three different areasper specimen were captured using either a 20× or 40× objective lens. Insome cases, a “combined score” or multiplicative index was derived thatincorporates both the percentage of positive cells and the stainingintensity according to the following formula: Combined score=(%positive)×(optical density score).

Example 31

This example demonstrates the ability of primary antibody-haptenconjugates to be visualized by fluorescent (Quantum Dot)immunohistochemistry (IHC).

Tonsil tissue sections were treated with an anti-Kappa polyclonalantibody (Dako) conjugated with haptens by the method in Examples 21,22, 23 or 24. The slides were developed using a standard protocol for anautomated stainer (BenchMark® XT, Ventana Medical Systems, Inc., Tucson,Ariz.). A typical automated protocol is as follows: the paraffin coatedtissue on the slide was heated to 75° C. for 8 minutes and treated twicewith EZPrep (VMSI), volume adjusted at 75° C. before application of theLiquid Cover Slip or LCS (VMSI). After two 8 minute incubation times at75° C., the slide was rinsed and EZPrep volume was adjusted, followedwith LCS to deparaffinize the tissue. The slide was cooled to 37° C.,incubated for 2 minutes and rinsed once with Reaction Buffer (VMSI). Theslide was treated with Cell Conditioner (VMSI) twice, followed by LCS.The slide was heated to 95° C. for 8 minutes, followed by LCS, and washeated to 100° C. for 4 minutes, followed by LCS. Cell Conditioner,incubate for 4 minutes, apply LCS, this incubation process with CellConditioner was repeated 9 times at 100° C. The slide was cooled downfor 8 minutes, rinsed with Reaction Buffer, volume adjusted, andfollowed by another dispense of LCS. The slide was heated to 37° C. for2 minutes and rinsed two times before the addition of anti-Kappa:haptenconjugate (100 μL at 1.0 mg/mL) followed by LCS and incubation at 37° C.for 32 minutes. The slide was rinsed twice with Reaction Buffer followedby the application of liquid cover slip and the addition ofQDot:anti-hapten conjugate (100 μL, 20-50 nmol) and incubated at 37° C.for 32 minutes. The slide was rinsed two times with buffer followed byLCS. The slide was removed from the instrument and treated to adetergent wash before manual application of a cover slip. Results wereinterpreted using a light microscope and aid in the differentialdiagnosis of pathophysiological processes, which may or may not beassociated with a particular antigen.

Example 32

This example demonstrates the ability of hapten-labeled DNA to bevisualized by chromogenic in situ hybridization (ISH). Automated silveror diaminobenzidine (DAB) in-situ hybridization protocols for detectionof HER2 gene copy number were developed on the Ventana Medical SystemsBenchmark XT instrument. Staining is completed on formalin fixedparaffin embedded tissue on glass slides within nine hours. The steps ofthe procedure are as follows: deparaffination, cell conditioning usingVMSI protease 3, addition of the hapten-labeled HER2 DNA probe (fromExample 26), tissue and probe denaturation, hybridization of four hours,and detection with chromogenic silver catalyzed by HRP. Specifically, aNitropyrazole labeled HER2 probe was hybridized in an automated fashionon formalin-fixed breast tissue, followed by detection withanti-Nitropyrazole Ab-HRP conjugate. Detection can be accomplished byuse of the UltraView detection kit (Ventana Medical Systems) or throughsilver deposition using the HER2 SISH (Ventana Medical Systems)automated protocol. Results were interpreted using a light microscopeand aid in the differential diagnosis of pathophysiological processes,which may or may not be associated with a particular antigen.

Example 33

This example demonstrates the ability of hapten-labeled DNA to bevisualized by fluorescent (Quantum Dot) in situ hybridization (ISH).Automated fluorescent (via Quantum Dots) in-situ hybridization protocolsfor detection of HER2 gene copy number were developed on the VentanaMedical Systems Benchmark XT instrument. Staining is completed onformalin fixed paraffin embedded tissue on glass slides within ninehours. The steps of the procedure are as follows: deparaffination, cellconditioning using VMSI protease 3, addition of the hapten-labeled HER2DNA probe (from Example 26), tissue and probe denaturation,hybridization of four hours, and detection with anti-hapten Ab:QuantumDot conjugates (from Example 29). Specifically, a Benzofurazan labeledHER2 probe was hybridized in an automated fashion, followed by detectionwith anti-Benzofurazan Ab-Quantum Dot 655 conjugate. Imaging wasperformed on a Nikon fluorescence scope.

Example 34

This example demonstrates the ability to multiplex primaryantibody-hapten conjugates and detect by multiplex fluorescent (QuantumDot) immunohistochemistry (IHC). This approach is schematicallyillustrated in FIG. 8. Tonsil tissue sections were treated with amixture of primary antibody-hapten conjugates: anti-CD20 Ab-biotin,anti-CD34 Ab-nitropyrazole, anti-CD45 Ab-thiazolesulfonamide, anti-KappaAb-dinitrophenyl, anti-Lambda Ab-rotenone, and anti-Ki67Ab-benzofurazan. The anti-CD20-biotin conjugate was made by couplingantibody thiol functional groups to maleimide-dPEG11-biotin used in a20-fold excess (described in Example 24). The anti-CD34-nitropyrazoleconjugate was formed by coupling to the Fc portion of the antibody usingpolyacrylamide hydrazide and a 20-fold excess of NHS-dPEG8-NP (describedin Example 22). This resulted in 12 nitropyrazole haptens per antibody.The anti-CD45-thiazolesulfonamide conjugate was made by reactingantibody lysines with a 20-fold excess of NHS-dPEG8-TS (described inExample 21). This resulted in 10 thiazolesulfonamide haptens perantibody. The Kappa-dinitrophenyl conjugate was formed by coupling tothe Fc portion of the antibody using polyacrylamide hydrazide and a100-fold excess of NHS-dPEG8-DNP (described in Example 22). Thisresulted in 62 dinitrophenyl haptens per antibody. Both the Lambdarotenone conjugate and the Ki67 benzofurazan conjugate were made byreacting antibody lysines to NHS-dPEG8-ROT and NHS-dPEG8-BF respectively(described in Example 21). This resulted in 0.3 rotenone haptens perantibody, and 2 benzofurazan haptens per antibody. The tonsil sectionswere then treated with a cocktail of secondary antibodies that weresynthesized using the procedure in Example 29: Gt α-biotin polyAb:QD 525(300 nM); Ms α-NP mAb:QD 655 (50 nM); Ms α-TS mAb:QD 565 (300 nM); Rbα-DNP polyAB:QD 605 (100 nM); Ms α-ROT mAb:QD 705 (200 nM); and Ms αBFmAb:QD 585 (300 nM).

Fluorescence Microscopy

Imaging was performed on a Nikon fluorescence scope. Unmixing offluorescence spectra was achieved utilizing a CRi camera. DAPI was usedfor counterstaining for the multiplexed tonsil sections.

FIG. 37 is multiplexed staining composite that was produced using amixture of primary antibody-hapten conjugates and sequentiallyvisualized with a mixture of anti-hapten antibody QDot conjugates asstated in FIGS. 31-36 and in this Example 34.

FIGS. 38-43 are images extracted from the multiplexed staining of FIG.37. FIG. 44 is a graph of wavelength versus relative fluorescence thatrepresents the wavelengths used to extract individual QDot signals fromthe multiplexed staining composite of FIG. 37. FIG. 44 also establishesthat the fluorescent signal is above the nominal autofluorescence of thetonsil tissue.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method for detecting a first target in a sample,comprising: contacting the sample with a first specific binding moietythat is conjugated to a first hapten; contacting the sample with a firstanti-hapten antibody specific to the first hapten; and detecting thefirst anti-hapten antibody; wherein the first hapten is sulfonamidehaving a formula

wherein R₁-R₄ are alkyl and R₂-R₃ are hydrogen; wherein the wavy lineattached to the nitrogen indicates attachment point of the first haptento the first specific binding moiety through an alkylene oxide linker,and wherein the first specific binding moiety binds specifically to thefirst target present in the sample.
 2. The method of claim 1, furthercomprising: contacting the sample with a second specific binding moietythat is conjugated to a second hapten; and then contacting the samplewith a second anti-hapten antibody specific to the second hapten; anddetecting the second anti-hapten antibody; wherein the second specificbinding moiety binds specifically to a second different target presentin the sample, and wherein the second hapten is selected from the groupconsisting of an oxazole, a pyrazole, a thiazole, a benzofurazan, atriterpene, a urea, a thiourea, a nitroaryl, a rotenoid, a coumarin, acyclolignan, a heterobiaryl, an azoaryl, and a benzodiazepine.
 3. Themethod of claim 2, wherein the second hapten is conjugated through asecond alkylene oxide linker.
 4. The method of claim 2, wherein thesecond hapten is a benzofurazan having a formula

where the R₅-R₈ substituents independently are selected from hydrogen,acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic,heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid,aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides,oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate,cyclic, cyano, ester, ether, exomethylene, halogen, heteroaryl,heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro,sulfhydryl, sulfonyl, sulfoxide, and combinations thereof, or two ormore of the R₅-R₈ substituents are atoms in a ring, at least one of theR₅-R₈ substituents being bonded to a linker or is a reactive groupsuitable for coupling to a linker or a carrier molecule.
 5. The methodof claim 4, wherein the benzofurazan is